Bacteria engineered to treat metabolic diseases

ABSTRACT

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of attenuating metabolic diseases are disclosed.

RELATED APPLICATIONS

The present application claims the benefit of priority to U.S. Provisional Patent Application No. 62/277,346, filed Jan. 11, 2016, U.S. Provisional Patent Application No. 62/293,695, filed Feb. 10, 2016, U.S. Provisional Patent Application No. 62/336,012, filed May 13, 2016, International Application No. PCT/US2016/032565, filed May 13, 2016, U.S. Provisional Patent Application No. 62/347,508, filed Jun. 8, 2016, U.S. Provisional Patent Application No. 62/347,554, filed Jun. 8, 2016, U.S. Provisional Patent Application No. 62/347,576, filed Jun. 8, 2016, U.S. Provisional Patent Application No. 62/348,416, filed Jun. 10, 2016, U.S. Provisional Patent Application No. 62/348,620, filed Jun. 10, 2016, U.S. Provisional Patent Application No. 62/354,681, filed Jun. 24, 2016, U.S. Provisional Patent Application No. 62/354,682, filed Jun. 24, 2016, International Application No. PCT/US2016/039444, filed Jun. 24, 2016, U.S. Provisional Patent Application No. 62/362,954, filed Jul. 15, 2016, U.S. Provisional Patent Application No. 62/385,235, filed Sep. 8, 2016, U.S. application Ser. No. 15/260,319, filed Sep. 9, 2016, International Application No. PCT/US2016/050836, filed Sep. 8, 2016, and U.S. Provisional Patent Application No. 62/423,170, filed Nov. 16, 2016, the contents of which are hereby incorporated by reference herein in their entirety.

BACKGROUND

Compositions and therapeutic methods for treating metabolic diseases are provided. In certain aspects, the compositions of the invention comprise bacteria that are genetically engineered to treat, modulate, and/or ameliorate metabolic diseases, particularly in low-oxygen environments, such as in the mammalian gut. In certain aspects, the compositions and methods of the invention as disclosed herein may be used for treating metabolic diseases such as obesity and type 2 diabetes. Obesity is caused by an imbalance between energy intake and expenditure, leading to the accumulation of unused energy in the form of fat. The World Health Organization considers obesity to be a global epidemic, and the United States Centers for Disease Control and Prevention estimates that nearly one third of adult Americans are obese. Diet and exercise may help reduce obesity and its associated pathologies, but adherence to a strict diet and exercise regime is challenging. Obesity may also be caused by other factors, e.g., mutations in genes regulating metabolic pathways (e.g., satiety, fatty acid oxidation, and mitochondrial function), which can contribute to energy imbalance. For example, congenital deficits in the signaling pathways for leptin, a satiety hormone, are known to cause obesity in humans and animal models.

Patients suffering from obesity are at increased risk of developing adverse physiological conditions, e.g., non-alcoholic fatty liver, cardiovascular diseases, type 2 diabetes mellitus (T2DM). The incidence of T2DM has increased 300% in the last three decades in the United States. T2DM patients are resistant to the effects of insulin, a hormone that regulates blood glucose levels, and frequently experience hyperglycemia, a condition in which blood glucose is above physiologically tolerable levels. When left untreated, hyperglycemia can result in severe complications such as hypertension, cardiovascular disease, inflammatory disease, blood vessel damage, nerve damage, cancer, and diabetes-induced coma.

T2DM involves the dysregulation of multiple metabolic organs, such as the pancreas, liver, skeletal muscle, adipose tissue, and brain, and it has been challenging to design therapeutics that target multiple tissue while avoiding systemic side effects. Insulin has been the first-line treatment for T2DM for decades. However, patients with severe T2DM may not respond to the insulin as a result of chronic insulin resistance. In addition, insulin must be administered multiple times throughout the day, which can adversely affect quality of life. Multiple therapies have been developed to treat T2DM, but not without limitations and sometimes life-threatening side effects. For example, thiazolidinedione was once widely used in order to increase the glucose metabolism in patients. However, the compound has been pulled from certain markets due to an increased association with heart failure (Nissen et al., 2007). Likewise, inhibitors of dipeptidyl peptidase-4 (DPP-4) have shown therapeutic promise, but may be linked to increased risk of pancreatic diseases (Karagiannis, et al., 2014).

Recently, researchers have demonstrated the close relationship between gut bacteria and metabolic disease (Harley et al., 2012). In obese mice, the ratio of firmicutes to bacteroidetes bacteria is increased (Harley et al., 2012; Mathur et al., 2015). These bacteria extract different amounts of energy from food, which may contribute to changes in energy balance. Similar changes have been also been observed in human studies (Harley et al., 2012; Mathur et al., 2015). Several molecules that are produced by gut bacteria have been shown to be metabolic regulators. For example, gut bacteria digest and break down dietary fiber into molecules such as acetate, butyrate, and propionate. These molecules are absorbed through intestinal cells, transferred to organs such as the liver and brain, and produce physiological changes, such as de novo glucose production and lipid synthesis (Brüssow et al., 2014; De Vadder et al., 2014; Lin et al., 2012). There has been an effort to engineer bacteria that produce N-acylphosphatidylethanolamines (NAPEs) (Chen et al., 2014). However, these bacteria express NAPEs constitutively and systemically, and NAPEs may be capable of “displac[ing] cholesterol from raft-like structures [and] may have dramatic implications for neural cell membrane function during stress and injury” (Terova et al., 2005). Thus, there is significant unmet need for effective, reliable, and/or long-term treatment for metabolic diseases, including obesity and T2DM.

SUMMARY

The disclosure provides genetically engineered bacteria that are capable of treating metabolic diseases, including but not limited to, type 2 diabetes, obesity-related symptoms, Nonalcoholic Steatohepatitis (NASH), Prader Willi Syndrome, and cardiovascular disorders. The genetically engineered bacteria comprise one or more gene(s) or gene cassette(s), for the production of molecules which, inter alia, act as metabolic and/or satiety effectors and/or modulators of the inflammatory status and/or are able convert excess bile salts into non-toxic molecules, as described herein.

Another aspect of the invention provides methods for selecting or targeting genetically engineered bacteria based on increased levels of metabolite consumption, or production of certain metabolites. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with metabolic disorders.

In some embodiments, the invention provides genetically engineered bacteria that are capable of producing one or more metabolic and/or satiety effector molecule(s), and/or one or more modulator(s) of inflammation, and/or one or more molecule(s) which reduces excess bile salt levels, and/or combinations thereof. In some embodiments, the invention provides genetically engineered bacteria that are capable of producing one or more metabolic and/or satiety effector molecule(s), and/or one or more modulator(s) of inflammation, and/or one or more molecule(s) which reduces excess bile salt levels, and/or combinations thereof, particularly in low-oxygen environments, e.g., the gut. In certain embodiments, the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to treat metabolic diseases. In certain embodiments, the metabolic and/or satiety effector molecule and/or modulator of inflammation or/and or effector of excess bile salt reduction is stably produced by the genetically engineered bacteria, and/or the genetically engineered bacteria are stably maintained in vivo and/or in vitro. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating metabolic diseases.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) or circuit(s), containing one or more native or non-native component(s), which mediate one or more mechanisms of action. The genetically engineered bacteria harbor these genes or gene cassettes or circuits on a plasmid or, alternatively, the genes/gene cassettes have been inserted into the chromosome at certain regions, where they do not interfere with essential gene expression. Additionally, one or more endogenous genes or regulatory regions within the bacterial chromosome may be mutated or deleted.

In some embodiments, the genetically engineered bacteria comprise one or more of the following: (1) one or more gene(s) or gene cassette(s) for the production of propionate, as described herein (2) one or more gene(s) or gene cassette(s) for the production of butyrate, as described herein (3) one or more gene(s) or gene cassette(s) for the production of acetate, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more of GLP-1 and GLP-1 analogs, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more bile salt hydrolases, as described herein (5) one or more gene(s) or gene cassette(s) for the production of tryptophan, as described herein; (6) one or more genes or gene cassettes for the production of a tryptophan metabolite, including an indole and/or indole metabolite, as described herein; (7) one or more genes for the production of one or more transporters, e.g. for the import of bile salts and/or metabolites, e.g. tryptophan and/or tryptophan metabolites, as described herein; (8) one or more polypetides for secretion, including but not limited to secretion of GLP-1 and its analogs, bile salt hydrolases, and tryptophan synthetic and/or catabolic enzymes of the tryptophan degradation pathways, and/or short chain fatty acid synthesis enzymes, in wild type or in mutated form (for increased stability or metabolic activity); (9) one or more components of secretion machinery, as described herein (10) one or more auxotrophies, e.g., deltaThyA; (11) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance; (12) one or more mutations/deletions to increase the flux through a metabolic pathway encoded by one or more genes or gene cassette(s), e.g. mutations/deletions in genes in NADH consuming pathways, genes involved in feedback inhibition of a metabolic pathway encoded by the gene(s) or gene cassette(s) genes, as described herein; and (13) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway.

These gene(s)/gene cassette(s) may be under the control of constitutive or inducible promoters. Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by molecules or metabolites indicative of liver damage (e.g., bilirubin) and/or metabolic disease, promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present in the gut, e.g., arabinose and tetracycline and othere described herein (e.g., metabolites not naturally present in the gut can be exogenously added). These gene(s)/gene cassette(s) may be under the control of constitutive and/or inducible promoters which are active or induced under in vitro conditions, e.g., during bacterial growth in a flask or other appropriate vessel for bacterial expansion, production, and/or manufacture, as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic of an E. coli that is genetically engineered to express a kynurenine biosynthesis cassette and/or a tryptophan biosynthesis cassette and/or tryptophan catabolic cassette which produces bioactive tryptophan metabolites described herein and/or GLP-1 and/or a propionate gene cassette and/or a butyrate gene cassette under the control of a FNR-responsive promoter and further comprising a secretion system and a metabolite transporter system.

FIG. 2A depicts a metabolic pathway for butyrate production FIGS. 2B and 2C depict two schematics of two different butyrate producing circuits (found in SYN-503 and SYN-504), both under the control of a tetracycline inducible promoter. FIG. 2D depicts a schematic of a third butyrate gene cassette (found in SYN-505) under the control of a tetracycline inducible promoter. SYN-503 comprises a bdc2 butyrate cassette under control of tet promoter on a plasmid. A “bdc2 cassette” or “bdc2 butyrate cassette” refres to a butyrate producing cassette that comprises at least the following genes: bcd2, etfB3, etfA3, hbd, crt2, pbt, and buk genes. SYN-504 comprises a ter butyrate cassette (ter gene replaces the bcd2, etfB3, and etfA3 genes) under control of tet promoter on a plasmid. A “ter cassette” or “ter butyrate cassette” refers to a butyrate producing cassette that comprises at least the following genes: ter, thiA1, hbd, crt2, pbt, buk. SYN-505 comprises a tesB butyrate cassette (ter gene is present and tesB gene replaces the pbt gene and the buk gene) under control of tet promoter on a plasmid. A “tes or tesB cassette or “tes or tesB butyrate cassette” refers to a butyrate producing cassette that comprises at least ter, thiA1, hbd, crt2, and tesB genes. An alternative butyrate cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiA1, hbd, crt2, and tesB genes. In some embodiments, the tes or tesB cassette is under control of an inducible promoter other than tetracycline. Exemplary inducible promoters which may control the expression of the tesB cassette include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

FIG. 3 depicts the gene organization of exemplary engineered bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIGS. 3A and 3B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 3A depicts relatively low butyrate production under aerobic conditions in which oxygen (O2) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk; black boxes) is expressed. FIG. 3B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIGS. 3C and 3D depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 3C, in the absence of NO, the NsrR transcription factor (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 3D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIGS. 3E and F depict the gene organization of an exemplary recombinant bacterium of the invention and its induction in the presence of H202. In FIG. 3E, in the absence of H2O2, the OxyR transcription factor (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 3F, in the presence of H2O2, the OxyR transcription factor interacts with H2O2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.

FIG. 4 depicts the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIGS. 4A and 4B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 4A depicts relatively low butyrate production under aerobic conditions in which oxygen (0₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, and buk; black boxes) is expressed. FIG. 4B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIGS. 4C and 4D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 4C, in the absence of NO, the NsrR transcription factor (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 4D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIGS. 4E and 4F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H₂O₂. In FIG. 4E, in the absence of H₂O₂, the OxyR transcription factor (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIGS. 4F, in the presence of H₂O₂, the OxyR transcription factor interacts with H₂O₂ and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.

FIG. 5 depicts the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIGS. 5A and 5B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 5A depicts relatively low butyrate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, and tesB; black boxes) is expressed. FIG. 5B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIGS. 5C and 5D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 5C, in the absence of NO, the NsrR transcription factor (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB; black boxes) is expressed. In FIG. 5D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIGS. 5E and 5F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H₂O₂. In FIG. 5E, in the absence of H₂O₂, the OxyR transcription factor (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB; black boxes) is expressed. In FIGS. 5F, in the presence of H₂O₂, the OxyR transcription factor interacts with H₂O₂ and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.

FIG. 6 depicts a graph of butyrate production using the circuits shown in FIG. 48. Cells were grown in M9 minimal media containing 0.2% glucose and induced with ATC at early log phase. As seen in FIG. 6A, similar amounts of butyrate were produced for each construct under aerobic vs anaerobic conditions. The ter strain produces more butyrate overall. pLogic031 comprises (bdc2 butyrate cassette under control of tet promoter on a plasmid) and pLogic046 comprises (ter butyrate cassette under control of tet promoter on a plasmid). FIG. 6B depicts butyrate production of pLogic046 (ter butyrate cassette under control of tet promoter on a plasmid)) and a Nissle strain comprising plasmid pLOGIC046-delta pbt.buk/tesB+, an ATC-inducible ter-comprising butyrate construct with a deletion in the pbt-buk genes and their replacement with the tesB gene. The tesB construct results in greater butyrate production.

FIG. 7 depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion. Strains depicted are SYN-503, SYN-504, SYN-510 (SYN-510 is the same as SYN-503 except that it further comprises a nuoB deletion), and SYN-511 (SYN-511 is the same as SYN-504 except that it further comprises a nuoB deletion). The NuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains. NuoB is a main protein complex involved in the oxidation of NADH during respiratory growth. In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production.

FIG. 8A depicts a schematic of a butyrate producing circuit under the control of an FNR promoter. FIG. 8B depicts a bar graph of anaerobic induction of butyrate production. FNR-responsive promoters were fused to butyrate cassettes containing either the bcd or ter circuits. Transformed cells were grown in LB to early log and placed in anaerobic chamber for 4 hours to induce expression of butyrate genes. Cells were washed and resuspended in minimal media w/ 0.5% glucose and incubated microaerobically to monitor butyrate production over time. SYN-501 led to significant butyrate production under anaerobic conditions.

FIG. 9 depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB-butyrate construct or the pLogic046-nsrR-norB-butyrate construct, which produce more butyrate as compared to wild-type Nissle.

FIG. 10 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H2O, 100 mM butyrate in H20, streptomycin resistant Nissle control or SYN501 comprising a PydfZ-ter ->pbt-buk butyrate plasmid. Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H2O2O (+) 200 mM butyrate.

FIG. 11 depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromsolmally integrated butyrate copies as compared to plasmid cpopies. Integrated butyrate strains, SYN1001 and SYN1002 gave comparable butyrate production to the plasmid strain SYN501.

FIG. 12 depicts a bar graph comparing butyrate concentrations produced in vitro by the butyrate cassette plasmid strain SYN501 as compared to Clostridia butyricum MIYARISAN (a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain) under aerobic and anaerobic conditions at the indicated timepoints. The Nissle strain comprising the butyrate cassette produces butyrate levels comparable to Clostridium spp. in RCM media.

FIG. 13 depicts a schematic illustrating a strategy for increasing butyrate and acetate production in engineered bacteria. Aerobic metabolism through the citric acid cycle (TCA cycle) (crossed out) is inactive in the anaerobic environment of the colon. E. coli makes high levels of acetate as an end production of fermentation. To improve acetate production, while still maintaining highlevels of butyrate production, targeted deletion can be introduced to prevent the production of unnecessary metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of competing routes (shown in in rounded boxes) are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions of interest therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.

FIG. 14A and FIG. 14B depict bar graphs showing Acetate/Butyrate production in 0.5% glucose MOPS (pH6.8) (FIG. 14A) and Acetate/Butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 14B). Deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were introduced into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes.

FIG. 15A and FIG. 15B depicts the gene organization of an exemplary engineered bacterium of the invention and its induction under low-oxygen conditions for the production of propionate. FIG. 15A depicts relatively low propionate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (pct, lcdA, lcdB, lcdC, e0, acrB, acrC; black boxes) are expressed. FIG. 15B depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate.

FIG. 16 depicts an exemplary propionate biosynthesis gene cassette.

FIG. 17A, FIG. 17B and FIG. 17C depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate. FIG. 17A depicts relatively low propionate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pd; black boxes) are expressed. FIG. 17B depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. FIG. 17C depicts an exemplary propionate biosynthesis gene cassette.

FIG. 18A, FIG. 18B and FIG. 18C depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate. FIG. 18A depicts relatively low propionate production under aerobic conditions in which oxygen (O₂) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pd, tesB; black boxes) are expressed. FIG. 18B depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate.

FIG. 19 depicts a schematic of an exemplary propionate biosynthesis gene cassette.

FIG. 20 depicts a schematic of an exemplary propionate biosynthesis gene cassette.

FIG. 21 depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production. Glucose and glycerol dissimilation pathways are shown under microaerobic conditions. In vivo, e.g., in a mammal, glycerol is not a substrate, and therefore only the glucose pathway is utilized.

FIG. 22 depicts a propionate production strategy. FIG. 22A a schematic of a construct comprising the sleeping beauty mutase operon from E. coli under the control of a heterologous FnrS promoter. FIG. 22B depicts a bar graph of proprionate concentrations produced in vitro by the wild type E coli BW25113 strain and a BW25113 strain which comprises the endogenous SBM operon under the control of the FnrS promoter, as depicted in the schematic in FIG. 22A.

FIG. 23 depicts a schematic of a construct comprising GLP-1 (1-37) under the control of the FliC promoter and 5′UTR containing the N-terminal flagellar secretion signal for secretion.

FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D depict schematics of the organization of exemplary GLP-1 secretion constructs with phoA (FIG. 24A and FIG. 24B) or OmpA (FIG. 24C and FIG. 24D) secretion tags. Three different RBS binding sites, 20K (FIG. 24A and FIG. 24C), 100K (FIG. 24B), and 67K (FIG. 24D) with varying strength (20<67<100) are used. In some embodiments, the Tet inducible promoter and the TetR sequence is replaced by a different inducible promoter system or a constitutive promoter in these constructs. In some embodiments, the background of the strain which contains these constructs and from which GLP-1 is secreted comprises a deletion or mutation in 1pp. FIG. 24A depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-20K RBS -PhoA-Glp1. FIG. 24B depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-100K RBS -PhoA-Glp1. FIG. 24C depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-20K RBS -OmpF-Glp1. FIG. 24D depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprisesTetR-pTet-67K RBS -OmpF-Glp1.

FIG. 25A and FIG. 25B depict schematics of the genetically engineered strains SYN2627 (comprising TetR-pTet-20K RBS -PhoA-G1p1) and SYN2643 (comprising TetR-pTet-20K RBS -PhoA-G1p1). Both strains comprise a deletion or mutation in 1pp. FIG. 25C depicts a bar graph showing the intracellular and secreted levels of GLP-1 as detected by ELISA assay for strains SYN2627 and SYN2643.

FIG. 26A and FIG. 26B depict line graphs of ELISA results. FIG. 26A depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA conducted on extracts from serum-starved Colo205 cells treated with supernatants from engineered bacteria comprising a PAL deletion and an integrated construct encoding hIL-22 with a phoA secretion tag. The data demonstrate that hIL-22 secreted from the engineered bacteria is functionally active. FIG. 26B depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA showing a antibody completion assay. Extracts from Colo205 cells were treated with the bacterial supernatants from the IL-22 overexpressing strain preincubated with increasing concentrations of neutralizing anti-IL-22 antibody. The data demonstrated that phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the hIL-22 antibody MAB7821.

FIG. 27 depicts bile salt metabolism. Bile salts are synthesized from cholesterol in the liver and stored in the gallbladder. After release into the duodenum, microbial bile salt hydrolase activity in the small intestine deconjugates the glycine or taurine molecules to produce primary bile acids (also known as unconjugated bile acids). Most bile acids are reabsorbed into the enterohepatic portal system, but some enter the large intestine where they are further metabolized by microbial 7α-dehydroxylase to produce secondary bile acids. Excess bile acids are also lost in the stool (200 mg-600 mg per day).

FIG. 28 depicts the structure of bile salts and the location at which bile salt hydrolase enzymes deconjugate the bile salts. BSH activity has been detected in Lactobacillus spp, Bifidobacterium spp, Enterococcus spp, Clostridium spp, and Bacteroides spp. BSH positive bacteria are gram positive with the exception of two Bacteroides strains. BSH in has been detected in pathogenic bacteria, e.g., Listeria monocytogenes and Enterococcus feacalis. E. coli does not demonstrate BSH actvity nor contain bsh homolog in genome

FIG. 29 depicts the state of one non-limiting embodiment of the bile salt hydrolase enzyme construct under inducing conditions. Expression of the bile salt hydrolase enzyme and a bile salt transporter are both induced by the FNR promoter in the absence of oxygen. The thyA gene has been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth. The recombinant bacterial cell may further comprise an auxotrophic mutation, a type III secretion system, and/or a kill switch, as further described herein.

FIG. 30 depicts schematic of the E. coli tryptophan synthesis pathway, including genes, enzymes, and reactions involved. The seven genes, or genetic segments, seven enzymes, or enzyme domains, and seven reactions, involved in tryptophan formation are shown. Only one of the reactions is reversible. The products of four other pathways contribute carbon and/or nitrogen during tryptophan formation. Two of the tryptophan pathway enzymes often function as polypeptide complexes: anthranilate synthase, consisting of the TrpG and TrpE polypeptides, and tryptophan synthase, consisting of the TrpB and TrpA polypeptides.

FIG. 31 depicts one embodiment of the disclosure in which the E. coli TRP synthesis enzymes are expressed from a construct under the control of a tetracycline inducible system.

FIG. 32 depicts a schematic of tryptophan metabolism in humans. The abbreviations for the enzymes are as follows: 3-HAO: 3-hydroxyl-anthranilate 3,4-dioxidase; AAAD: aromatic-amino acid decarboxylase; ACMSD, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase; HIOMT, hydroxyl-O-methyltransferase; IDO, indoleamine 2,3-dioxygenase; KAT, kynurenine amino transferases I-III; KMO: kynurenine 3-monooxygenase; KYNU, kynureninase; NAT, N-acetyltransferase; TDO, tryptophan 2,3-dioxygenase; TPH, tryptophan hydroxylase; QPRT, quinolinic acid phosphoribosyl transferase. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the tryptophan metabolism enzymes depicted in FIG. 32, or bacterial functional homologs thereof. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes which produce one or more of the tryptophan metabolites depicted in FIG. 32. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments, the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

FIG. 33 depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IPA) and Indole-3-aldehyde (I3A), in the gut lumen. IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function. I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL-22 production. Activation of AhR plays a crucial role in gut immunity, such as in maintaining the epithelial barrier function and promoting immune tolerance to promote microbial commensalism while protecting against pathogenic infections. Indole has a number of roles, such as a signaling molecule to intestinal L cells to produce glucagon-like protein 1 (GLP-1) or as a ligand for AhR (Zhang et al. Genome Med. 2016; 8: 46).

FIG. 34 depicts a schematic of the trypophan metabolic pathway. Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled, respectively (see, e.g., Lamas et al., CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands; Nature Medicine 22, 598-605 (2016). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in FIG. 34. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 34, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In certain embodiments the one or more cassettes are under the control of constitutive promoters.

FIG. 35A depicts a schematic of the bacterial tryptophan metabolism, as described, e.g., in Enzymes are numbered as follows 1) Trp 2,3 dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3) kynureninase (EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC 2.6.1.27); 6) indole lactate dehydrogenase (EC1.1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13) Trp 2-monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0). The dotted lines (-) indicate a spontaneous reaction. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIG. 35. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 35. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments, the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In certain embodiments the one or more cassettes are under the control of constitutive promoters. FIG. 35B Depicts a schematic of tryptophan derived pathways. Known AHR agonists are with asterisk. Abbreviations are as follows. Trp: Tryptophan; TrA: Tryptamine; IAAld: Indole-3-acetaldehyde; IAA: Indole-3-acetic acid; FICZ: 6-formylindolo(3,2-b)carbazole; IPyA: Indole-3-pyruvic acid; IAM: Indole-3-acetamine; IAOx: Indole-3-acetaldoxime; IAN: Indole-3-acetonitrile; N-formyl Kyn: N-formylkynurenine; Kyn:Kynurenine; KynA: Kynurenic acid; I3C: Indole-3-carbinol; IAld: Indole-3-aldehyde; DIM: 3,3′-Diindolylmethane; ICZ: Indolo(3,2-b)carbazole.

FIG. 36A, FIG. 36B, FIG. 36C, and FIG. 36D depicts schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA; thymidine dependence). FIG. 36A shows a schematic depicting an exemplary Tryptophan circuit. Tryptophan is produced from its precursor, chorismate, through expression of the trpE, trpG-D (also referred to as trpD), trpC-F (also referred to as trpC), trpB and trpA genes. Optional knockout of the tryptophan repressor trpR is also depicted. Optional production of chorismate through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36B, and/or FIG. 36C, and/or FIG. 36D. FIG. 36B depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 36A and/or described in the description of FIG. 36A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36C, and/or FIG. 36D. Optionally, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. FIG. 36C depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further comprises either a wild type or a feedback resistant SerA gene. Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD1 to NADH. E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 36A and/or described in the description of FIG. 36A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36B, and/or FIG. 36D. Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. FIG. 36D depicts a non-limiting example of a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further optionally comprises either a wild type or a feedback resistant SerA gene. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 36A and/or described in the description of FIG. 36A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 36B, and/or FIG. 36C. Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. Optionally, the bacteria may also comprise a deletion in PheA, which prevents conversion of chorismate into phenylalanine and thereby promotes the production of anthranilate and tryptophan. FIG. 37A, FIG. 37B, FIG. 37D, FIG. 37D, FIG. 37E, FIG. 37F, FIG. 37G, and FIG. 37H depict schematics of non-limiting examples of embodiments of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included. FIG. 37A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. In certain embodiments the one or more cassettes are under the control of inducible promoters. In certain embodiments the one or more cassettes are under the control of constitutive promoters. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for Tryptophan decarboxylase, e.g., from Catharanthus roseus, which converts tryptophan to tryptamine, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37B depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli , or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or sta0 (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37C depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA (Monoamine oxidase, e.g., from E. coli ), which converts tryptophan to indole-3-acetaldehyde and FICZ, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37D depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for cyp79B2, (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), which together convert tryptophan to indole-3-acetonitrile, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37E depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) which together convert tryptophan to kynurenine, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37F depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g.,from homo sapiens or AADAT (Kynurenine/alpha-amino adipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens, which together produce kynureninic acid from tryptophan, under the control of an inducible promoter, e.g., an FNR promoter. FIG. 37G depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 36A and/or and/or FIG. 36B, and/or FIG. 36C, and/or FIG. 36D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for tnaA (tryptophanase, e.g., from E. coli ), which converts tryptophan to indole, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 37H depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. The genetically engineered bacteria comprise a circuit comprising pne2 (myrosinase, e.g., from Arabidopsis thaliana) under the control of an inducible promoter, e.g. an FNR promoter. The engineered bacterium shown in any of FIG. 37A, FIG. 37B, FIG. 37D, FIG. 37D, FIG. 37E, FIG. 37F, FIG. 37G and FIG. 37H may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.

FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, and FIG. 38E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid. In certain embodiments, the one or more cassettes are under the control of inducible promoters. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. In FIG. 38A, the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli , or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana) which together produce indole-3-acetic acid from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38B the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g.,from Catharanthus roseus and/or Clostridium sporogenes) of tynA (Monoamine oxidase, e.g., from E. coli ) and or iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana), e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38C the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli , or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 (indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana) e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38D the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IaaM (Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 38E the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana and cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana) and nitl (Nitrilase, e.g., from Arabidopsis thaliana) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an FNR promoter. the engineered bacterium shown in any of FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, and FIG. 38E may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth. In FIG. 38F the optional circuits for tryptophan production are as depicted and described in FIG. 36A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 36B and/or FIG. 36C and/or FIG. 36D. Alternatively, optionally, tryptophan can be imported through a transporter. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.

FIG. 39A, FIG. 39B, and FIG. 39C depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA; thymidine dependence). FIG. 39A a depicts non-limiting example of a tryptamine producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine. FIG. 39B depicts a non-limiting example of an indole-3-acetate producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate. FIG. 39C depicts a non-limiting example of an indole-3-propionate-producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises a circuit as described in FIG. 44, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indol-3y1)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH/ and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3-yl)pyruvate into indole-3-lactate).

FIG. 40A and FIG. 40B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production. FIG. 40A depicts a schematic showing intermediates in tryptophan biosynthesis and the gene products catalyzing the production of these intermediates. Phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) are used to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). DHAP is catabolized to chorismate and then anthranilate, which is converted to tryptophan (Trp) by the tryptophan operon. Alternatively, chorismate can be used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe). In the serine biosynthesis pathway, D-3-phosphoglycerate is converted to serine, which can also be a source for tryptophan biosynthesis. AroG AroF, AroH: DAHP synthase catalyzes an aldol reaction between phosphoenolpyruvate and D-erythrose 4-phosphate to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). There are three isozymes of DAHP synthase, each specifically feedback regulated by tyrosine (AroF), phenylalanine (AroG) or tryptophan(AroH). AroB: Dehydroquinate synthase (DHQ synthase) is involved in the second step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ synthase catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to dehydroquinate (DHQ). AroD: 3-Dehydroquinate dehydratase (DHQ dehydratase) is involved in the 3rd step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ dehydratase catalyzes the conversion of DHQ to 3-dehydroshikimate and introduces the first double bond of the aromatic ring. AroE, YdiB: E. coli expresses two shikimate dehydrogenase paralogs, AroE and YdiB. Shikimate dehydrogenase is involved in the 4th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme converts 3-dehydroshikimate to shikimate by catalyzing the NADPH linked reduction of 3-dehydro-shikimate. AroL/AroK: Shikimate kinase is involved in the fifth step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. Shikimate kinase catalyzes the formation of shikimate 3-phosphate from shikimate and ATP. There are two shikimate kinase enzymes, I (AroK) and II (AroL). AroA: 3-Phosphoshikimate-1-carboxyvinyltransferase (EPSP synthase) is involved in the 6th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. EPSP synthase catalyzes the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate to the hydroxyl group of carbon 5 of shikimate 3-phosphate with the elimination of phosphate to produce 5-enolpyruvoyl shikimate 3-phosphate (EPSP). AroC: Chorismate synthase (AroC) is involved in the 7th and last step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate into chorismate, which is the branch point compound that serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis. This reaction introduces a second double bond into the aromatic ring system. TrpEDCAB (E coli trp operon): TrpE (anthranilate synthase) converts chorismate and L-glutamine into anthranilate, pyruvate and L-glutamate. Anthranilate phosphoribosyl transferase (TrpD) catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5′-phosphoribosyl)-anthranilate. The phosphoribosyl transferase and anthranilate synthase contributing portions of TrpD are present in different portions of the protein. Bifunctional phosphoribosylanthranilate isomerase/indole-3-glycerol phosphate synthase (TrpC) carries out the third and fourth steps in the tryptophan biosynthesis pathway. The phosphoribosylanthranilate isomerase activity of TrpC catalyzes the Amadori rearrangement of its substrate into carboxyphenylaminodeoxyribulose phosphate. The indole-glycerol phosphate synthase activity of TrpC catalyzes the ring closure of this product to yield indole-3-glycerol phosphate. The TrpA polypeptide (TSase α) functions as the a subunit of the tetrameric (α2-β2) tryptophan synthase complex. The TrpB polypeptide functions as the 0 subunit of the complex, which catalyzes the synthesis of L-tryptophan from indole and L-serine, also termed the β reaction. TnaA: Tryptophanase or tryptophan indole-lyase (TnaA) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the cleavage of L-tryptophan to indole, pyruvate and NH4+. PheA: Bifunctional chorismate mutase/prephenate dehydratase (PheA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis. TyrA: Bifunctional chorismate mutase/prephenate dehydrogenase (TyrA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in tyrosine biosynthesis. TyrB, ilvE, AspC: Tyrosine aminotransferase (TyrB), also known as aromatic-amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis. TyrB catalyzes the transamination of 2-ketoisocaproate, p-hydroxyphenylpyruvate, and phenylpyruvate to yield leucine, tyrosine, and phenylalanine, respectively. TyrB overlaps with the catalytic activities of branched-chain amino-acid aminotransferase (IlvE), which also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC), which also produces phenylalanine. SerA: D-3-phosphoglycerate dehydrogenase catalyzes the first committed step in the biosynthesis of L-serine. SerC: The serC-encoded enzyme, phosphoserine/phosphohydroxythreonine aminotransferase, functions in the biosythesis of both serine and pyridoxine, by using different substrates. Pyridoxal 5′-phosphate is a cofactor for both enzyme activities. SerB: Phosphoserine phosphatase catalyzes the last step in serine biosynthesis. Steps which are negatively regulated by the Trp Repressor (2), Tyr Repressor (1), or tyrosine (3), phenylalanine (4), or tryptophan (4) or positively regulated by trptophan (6) are indicated. FIG. 40B depicts a schematic showing exemplary engineering strategies which can improve tryptophan production. Each of these exemplary strategies can be used alone or two or more strategies can be combined to increase tryptophan production. Intervention points are in bold, italics and underlined. In one embodiment of the disclosure, bacteria are engineered to express a feedback resistant from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express AroL. In one embodiment, bacteria are engineered to comprise one or more copies of a feedback resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered to comprise one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEtbr, and/or TrpD, and/or TrpC, and/or TrpA, and/or TrpB. In one embodiment, endogenous TnaA is knocked out through mutation(s) and/or deletion(s). In one embodiment, bacteria are engineered to comprise one or more additional copies of SerA. In one embodiment, bacteria are engineered to comprise one or more additional copies of YddG, a tryptophan exporter. In one embodiment, endogenous PheA is knocked out through mutation(s) and/or deletion(s). In one embodiment, two or more of the strategies depicted in the schematic of FIG. 40B are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed.

FIG. 41A and FIG. 41B and FIG. 41C depict bar graphs showing tryptophan production by various engineered bacterial strains. FIG. 41A depicts a bar graph showing tryptophan production by various tryptophan producing strains. The data show expressing a feedback resistant form of AroG (AroG^(fbr)) is necessary to get tryptophan production. Additionally, using a feedback resistant trpE (trpE^(fbr)) has a positive effect on tryptophan production. FIG. 41B shows tryptophan production from a strain comprising a tet-trpE^(fbr)DCBA, tet-aroG^(fhr) construct, comparing glucose and glucuronate as carbon sources in the presence and absence of oxygen. It takes E. coli two molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan. When glucose is used as the carbon source, 50% of all available PEP is used to import glucose into the cell through the PTS system (Phosphotransferase system). Tryptophan production is improved by using a non-PTS sugar (glucuronate) aerobically. The data also show the positive effect of deleting tnaA (only at early time point aerobically). FIG. 41C depicts a bar graph showing improved tryptophan production by engineered strain comprising ΔtrpRΔtnaA, tet-trpE^(fbr)DCBA, tet-aroG^(fbr) through the addition of serine.

FIG. 42 depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476. SYN2126 ΔtrpRΔtnaA. ΔtrpRΔtnaA, tet-aroGfbr. SYN2339 comprises ΔtrpRΔtnaA, tet-aroGfbr, tet-trpEtbrDCBA. SYN2473 comprises ΔtrpRΔtnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA. SYN2476 comprises ΔtrpRΔtnaA, tet-trpEtbrDCBA. Results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production.

FIG. 43 depicts a schematic of an indole-3-propionic acid (IPA) synthesis circuit. IPA produced by the gut microbiota has a significant positive effect on barrier integrity. IPA does not signal through AhR, but rather through a different receptor (PXR) (Venkatesh et al., Symbiotic Bacterial Metabolites Regulate Gastrointestinal Bardrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, Aug. 21, 2014). In some embodiments, IPA can be produced in a synthetic circuit by expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus) and indole-3-acrylate reductase (e.g., from Clostridum botulinum). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

FIG. 44 depicts a schematic of indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis(TrA) circuits. Enzymes are as follows : 1. TrpDH: tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108; FldH1/FldH2: indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole-3-lactate dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; AcuI: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. 1pdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae; 1ad1: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.

Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH₃, NAD(P)H and H⁺. Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indol-3yl)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (FldA) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (F1dD) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate decarboxylase (1pdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAAld) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAAld) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

FIG. 45 depicts a bar graph showing tryptophan and indole acetic acid production for strains SYN2126, SYN2339 and SYN2342. SYN2126: comprises ΔtrpR and ΔtnaA (ΔtrpRΔtnaA). SYN2339 comprises circuitry for the production of tryptophan (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises ipdC-iad1 incorporated at the end of the second construct (AtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iad1 (p15A)). SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 converts all trypophan it produces into IAA.

FIG. 46 depicts a bar graph showing tryptophan and tryptamine production for strains SYN2339, SYN2340, and SYN2794. SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine and comprises ΔtrpRΔtnaA, tetR-P_(tet)-trpE^(fbr)DCBA (pSC101), tetR-P_(tet)aroG^(fhr) (p15A). SYN2340 comprises ΔtrpRΔtnaA, tetR-P_(tet)-trpE^(fbr)DCBA (pSC101), tetR-P_(tet)aroG^(fbr)-tdc_(Cr) (p15A). SYN2794 comprises ΔtrpRΔtnaA, tetR-P_(tet)-trpE^(fbr)DCBA (pSC101), tetR-P_(tet)aroG^(fbr)-tdc_(Cs) (p15A). Results indicate that Tdc_(Cs) from Clostridium sporogenes is more efficient the Tdc_(Cr) from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine.

FIG. 47 depicts a schematic of an E. coli that is genetically engineered to express a butyrate cassette.

FIG. 48 depicts a schematic of an E. coli that is genetically engineered to express a a propionate biosynthestic cassette.

FIG. 49 depicts a schematic of an E. coli that is genetically engineered to express a GLP-1 and a secretion system as known in the art or described herein.

FIG. 50 depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Kynurenine biosynthetic cassette is expressed to produce Kynurenine. Both the transporter and Kynurenine biosynthetic cassette genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA).

FIG. 51 depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Tryptophan is synthesized and then Kynurenine is synthesized from the synthesized tryptophan or from tryptophan imported into the cell. Both the transporter and kynureninase biosynthetic genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA).

FIG. 52 depicts a schematic of an E. coli that is genetically engineered to express a butyrate and a propionate biosynthestic cassette.

FIG. 53 depicts a schematic of an E. coli that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to kynurenine or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. Export mechanism for kynurenine and/or tryptophan is also expressed or provided.

FIG. 54 depicts a schematic of an E. coli that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to tryptamine and/or indole acetic acid or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. A tryptophan transporter for import of tryptophan also expressed. Export mechanism for kynurenine is also expressed or provided.

FIG. 55 depicts a schematic of an E. coli that is genetically engineered to produce butyrate, tryptophan metabolites, and tryptophan (which can be converted to bioactive tryptophan metabolites or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. Export mechanism for tryptophan and/or tryptophan metabolites is also expressed or provided.

FIG. 56 depicts a schematic of an E. coli that is genetically engineered to produce butyrate, and propionate, kynurenine and/or other tryptophan metabolites, and GLP-1, under the control of a FNR-responsive promoter and further comprising a secretion system, e.g., for GLP-1 secretion as known in the art or described herein. Export mechanism for kynurenine/or tryptophan metabolites is also expressed or provided.

FIG. 57 depicts a map of exemplary integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites. The malE/K site is circled. In some embodiments of the disclosure, FNR-ArgAfbr is inserted at the malEK locus.

FIG. 58 depicts three bacterial strains which constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light. Unmodified E. coli Nissle (strain 4) is non-fluorescent.

FIG. 59 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (MoAs).

FIG. 60 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple MoAs. In some embodiments, an Glp-1 expression circuit, a butyrate production circuit, a propionate production circuit, and a tryptophan and/or indole metabolite biosynthetic cassette are inserted at four or more different chromosomal insertion sites

FIG. 61 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

FIG. 62 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The beta-domain is recruited to the Bam complex where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. The therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.

FIG. 63 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP-binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

FIG. 64 depicts a schematic of the outer and inner membranes of a gram-negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the extracellular space, e.g., therapeutic polypeptides of eukaryotic origin containing disulphide bonds. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype.

FIG. 65 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen. An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell. An inducible promoter (small arrow, bottom), e.g. a FNR-inducible promoter, drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons).

FIG. 66A, FIG. 66B, and FIG. 66C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., metabolic and/or satiety effector and/or immune modulator polypeptides described herein, which are secreted using components of the flagellar type III secretion system. A therapeutic polypeptide of interest, is assembled behind a fliC-5′UTR, and is driven by the native fliC and/or fliD promoter (FIG. 66A and FIG. 66B) or a tet-inducible promoter (FIG. 66C). In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose can be used. In certain embodiments the one or more cassettes are under the control of constitutive promoters. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD). Optionally, an N terminal part of FliC is included in the construct, as shown in FIG. 66B and FIG. 66D.

FIG. 67A and FIG. 67B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., metabolic and/or satiety effector and/or immune modulator polypeptides described herein, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpl. Optionally, periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a tet promoter (FIG. 67A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, FIG. 67B), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. In certain embodiments the one or more cassettes are under the control of constitutive promoters.

FIG. 68 depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (P_(araBAD)), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. FIG. 68A also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell. FIG. 68B depicts a non-limiting embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit. FIG. 68C depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. The araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.

FIG. 69 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

FIG. 70 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.

FIG. 71 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death.

FIG. 72 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

FIG. 73 depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-316.

FIG. 61A, FIG. 74B, FIG. 74C, and FIG. 74D depict schematics of non-limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (FIG. 74A and FIG. 74B), which also contains a chromosomal component (shown in FIG. 74C and FIG. 74D). The Biosafety Plasmid System Vector comprises Kid Toxin and R6K minimal ori, dapA (FIG. 74A) and thyA (FIG. 74B) and promoter elements driving expression of these components. In some embodiments, bla is knocked out and replaced with one or more constructs described herein, and one or more metabolic and/or satiety effector(s) and/or immune modulator are expressed from an inducible or constitutive promoter. FIG. 74C and FIG. 74D depict schematics of the gene organization of the chromosomal component of a biosafety system. FIG. 74C depicts a construct comprising low copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a low copy RBS containing promoter. FIG. 74D depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a medium copy RBS containing promoter. If the plasmid containing the functional DapA is used (as shown in FIG. 74A), then the chromosomal constructs shown in FIG. 74C and FIG. 74D are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in FIG. 74B), then the chromosomal constructs shown in FIG. 74C and FIG. 74D are knocked into the ThyA locus. In this system, the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid.

FIG. 75 depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown in Table 2 (Pfnr1-5). Different FNR-responsive promoters were used to create a library of anaerobic-inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+O₂) or anaerobic conditions (−O₂). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β-galactosidase colorimetric assays.

FIG. 76A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P_(fnrs)). LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. FIG. 76B depicts FNR promoter activity as a function of (3-galactosidase activity in SYN340. SYN340, an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high-level gene expression within 1 hr under anaerobic conditions. FIG. 76C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.

FIG. 77A and FIG. 77B depict schematics of ATC (FIG. 77A) or nitric oxide-inducible (FIG. 77B) reporter constructs. These constructs, when induced by their cognate inducer, lead to expression of GFP. Nissle cells harboring plasmids with either the control, ATC-inducible P_(tet)-GFP reporter construct or the nitric oxide inducible P_(nsrR)-GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units. FIG. 77C depicts a schematic of the constructs. FIG. 77D depicts a dot blot of bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter. DSS-treated mice serve as exemplary models for HE. As in HE subjects, the guts of mice are damaged by supplementing drinking water with 2-3% dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated promoters induced in DSS-treated mice.

FIG. 78A depicts a “Oxygen bypass switch” useful for aerobic pre-induction of a strain comprising one or proteins of interest (POI), e.g., one or more metabolic and/or satiety effector(s) (POI1) and /or immune modulator and/or one or more transporter(s)/importer(s) and/or exporter(s) (PGI2) under the control of a low oxygen FNR promoter in vitro in a culture vessel (e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture). In some embodiments, it is desirable to pre-load a strain with active payload(s) prior to administration. This can be done by pre-inducing the expression of these enzymes as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration. In some embodiments, strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more proteins of interest. In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic or microaerobic conditions with one or more proteins of interest. This allows more efficient growth and, in some cases, reduces the build-up of toxic metabolites.

FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis A J, The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo.

In some embodiments, a Lad promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

FIG. 78B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of FNRS24Y. By using a ribosome binding site optimization strategy, the levels of FnrS24Y expression can be fine-tuned, e.g., under optimal inducing conditions (adequate amounts of arabinose for full induction). Fine-tuning is accomplished by selection of an appropriate RBS with the appropriate translation initiation rate. Bioinformatics tools for optimization of RBS are known in the art.

FIG. 78C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy. Bioinformatics tools for optimization of RBS are known in the art. In one strategy, arabinose controlled POI genes can be integrated into the chromosome to provide for efficient aerobic growth and pre-induction of the strain (e.g., in flasks, fermenters or other appropriate vesicles), while integrated versions of PfnrS-POI constructs are maintained to allow for strong in vivo induction.

FIG. 79 depicts a construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.

FIG. 80 depicts the gene organization of an exemplary construct, comprising a cloned protein of interest (POI) gene under the control of a Tet promoter sequence and a Tet repressor gene.

FIG. 81 depicts the gene organization of an exemplary construct comprising Lad in reverse orientation, and a IPTG inducible promoter driving the expression of a protein of interest (POI, e.g., one or more metabolic effector(s) described herein). In some embodiments, this construct is useful for pre-induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG. In some embodiments, this construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs. In some embodiments, the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting.

FIG. 82A, FIG. 82B, and FIG. 82C depict schematics of non-limiting examples of constructs expressing a protein of interest (POI). FIG. 82A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter. The construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI. The temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees C. but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, the construct comprises SEQ ID NO: 101. In some embodiments, this construct is used alone. In some embodiments, the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In some embodiments, the construct provides in vivo activity. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with other POI constructs, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. In some embodiments, a temperature sensitive system can be used to set up a conditional auxotrophy. In a a strain comprising deltaThyA or deltaDapA, a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower).

FIG. 82B depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of a rhamnose inducible promoter. For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a bio safety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

FIG. 82C depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of an arabinose inducible promoter. The arabinose inducible POI construct comprises AraC (in reverse orientation), a region comprising an Arabinose inducible promoter, and the POI gene. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POI(s) prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a bio safety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

FIG. 83A depicts a schematic of the gene organization of a PssB promoter. The ssB gene product protects ssDNA from degradation; SSB interacts directly with numerous enzymes of DNA metabolism and is believed to have a central role in organizing the nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination and repair. The PssB promoter was cloned in front of a LacZ reporter and beta-galactosidase activity was measured. FIG. 83B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1:100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. Thus, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. In one non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA—as the case may be—are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch).

FIG. 84 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

FIG. 85 depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 109 CFU, and at each timepoint, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each compartment were determined by serial dilution plating. FIG. 85 depicts a bar graph of residence over time for streptomycin resistant Nissle.

FIG. 86 depicts a schematic diagram of a wild-type clbA construct (upper panel) and a schematic diagram of a clbA knockout construct (lower panel).

FIG. 87 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animla disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen.

FIG. 88A, B, C, D, and E depict a schematic of non-limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure. FIG. 88A depicts the parameters for starter culture 1 (SC1): loop full—glycerol stock, duration overnight, temperature 37° C., shaking at 250 rpm. FIG. 88B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37° C., shaking at 250 rpm. FIG. 88C depicts the parameters for the production bioreactor: inoculum—SC2, temperature 37° C., pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300-1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours. FIG. 88D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash 1×10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS. FIG. 88E depicts the parameters for vial fill/storage: 1-2 mL aliquots, −80° C.

DESCRIPTION OF EMBODIMENTS

The invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating metabolic diseases. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s), or a gene cassette(s) encoding one or more non-native biosynthetic pathway(s) for producing one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s), or a gene cassette(s) encoding one or more non-native biosynthetic pathway(s) for producing one or more non-native metabolic and/or satiety effector and/or immune modulator molecule(s) and further comprise gene sequence encoding one or more immune modulator molecules, such as any of the immune modulators described herein. In some embodiments, the gene sequence or gene cassette is further operably linked to an inducible promoter, for example, a regulatory region that is controlled by a transcription factor that is capable of sensing low-oxygen conditions, inflammatory conditions, or other tissue-specific or environment-specific conditions. In certain embodiments, the genetically engineered bacteria are capable of producing metabolic and/or satiety effector molecule and/or anti-inflammatory molecules in low-oxygen environments, e.g., the gut. Thus, the genetically engineered bacteria and pharmaceutical compositions comprising those bacteria may be used in order to treat and/or prevent conditions associated with metabolic diseases, including obesity and type 2 diabetes.

In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

As used herein, “metabolic diseases” include, but are not limited to, type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome.

Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility.

Metabolic diseases are associated with a variety of physiological changes, including but not limited to elevated glucose levels, elevated triglyceride levels, elevated cholesterol levels, insulin resistance, high blood pressure, hypogonadism, subfertility, infertility, abdominal obesity, pro-thrombotic conditions, and pro-inflammatory conditions. A metabolic effector is a molecule that is capable of minimizing any one or more of said physiological changes. For example, a metabolic effector molecule may enhance the body's sensitivity to insulin, thereby ameliorating insulin resistance. Insulin resistance is a physiological condition in which the body's insulin becomes less effective at lowering blood sugar. Excess blood sugar can cause adverse health effects such as type 2 diabetes. “Satiety” is used to refer to a homeostatic state in which a subject feels that hunger or food craving is minimized or satisfied. A satiety effector is a molecule that contributes to the minimization or satisfaction of said hunger or food craving. A molecule may be primarily a metabolic effector or primarily a satiety effector. A molecule may be both a metabolic and satiety effector, e.g., GLP-1.

“Metabolic effector molecules” and/or “satiety effector molecules” include, but are not limited to, n-acyl-phophatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, entero statin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA). Such molecules may also include compounds that inhibit a molecule that promotes metabolic disease, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor. A metabolic and/or satiety effector molecule may be encoded by a single gene, e.g., glucogon-like peptide 1 is encoded by the GLP-1 gene. Alternatively, a metabolic and/or satiety effector molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., propionate. These molecules may also be referred to as therapeutic molecules.

An “anti-inflammatory” or anti-inflammatory molecule” refers to a molecule that reduces, decreases, inhibits, or prevents an inflammatory response, either directly or indirectly. Non-limiting examples of anti-inflammatory molecules include short-chain fatty acids (e.g., butyrate, propionate, acetate), certain tryptophan metabolites, e.g., indoles and indole metabolites, as described herein, certain cytokines, including but not limited to, IL-10, IL-22, IL-4, IL-13, IFNa, and TGFB.

An “immune modulator” or “immune modulator molecule” refers to a molecule that modulates an inflammatory response. Non-limiting examples of immune modulator molecules include molecules that directly modulate an inflammatory response and also includes molecules that activate (stimulate or increase the activity of) or inhibit (decrease the activity of) molecules that directly modulate an inflammatory response. For example, an immune modulator can decrease levels of inflammatory growth factors and cytokines, e.g., IL-1β, IL-6, and/or TNF-α and proinflammatory signaling, e.g. NF-kappaB signaling and/or can increase levels of anti- inflammatory growth factors and cytokines, e.g., IL4, IL-10, IL-13, IFN-alpha and/or transforming growth factor-beta. Other immune modulators include, but are not limited to, short-chain fatty acids (e.g., butyrate, propionate, acetate), certain tryptophan metabolites, e.g., indoles and indole metabolites, as described herein, certain cytokines, including but not limited to, IL-10, IL-22, IL-4, IL-13, IFNa, and TGFB.

As used herein, the term “engineered bacterial cell” or “engineered bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, an engineered bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Engineered bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

A “programmed bacterial cell” or “programmed engineered bacterial cell” is an engineered bacterial cell that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed.

As used herein, a “heterologous” gene or “heterologous sequence” refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell. “Heterologous gene” includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism. As used herein, the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.

As used herein, the term “coding region” refers to a nucleotide sequence that codes for a specific amino acid sequence. The term “regulatory sequence” refers to a nucleotide sequence located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter.

As used herein, a “gene cassette” or “operon” or “genetic circuit” encoding a biosynthetic pathway or catabolic pathway refers to the two or more genes that are required to produce a metabolic and/or satiety effector and/or immune modulator molecule, e.g., propionate and/or immune modulator molecule (e.g., tryptophane metabolite, e.g., indole). In addition to encoding a set of genes capable of producing said molecule, the gene cassette or operon or “genetic circuit” may also comprise additional transcription and translation elements, e.g., a ribosome binding site.

A “butyrogenic gene cassette,” “butyrate biosynthesis gene cassette,” and “butyrate operon” are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway. Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. The genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria. A butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013). One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. A butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiA1 from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. The butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB.

Likewise, a “propionate gene cassette” or “propionate operon” refers to a set of genes capable of producing propionate in a biosynthetic pathway. Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. The genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria. In some embodiments, the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and Kandasamy 2012 Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation). This operon catalyses the reduction of lactate to propionate. Dehydration of (R)-lactoyl-CoA leads to the production of the intermediate acryloyl-CoA by lactoyl-CoA dehydratase (LcdABC). Acrolyl-CoA is converted to propionyl-CoA by acrolyl-CoA reductase (EtfA, AcrBC). In some embodiments, the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC, are replaced by the acuI gene from R. sphaeroides. This gene product catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides; Asao 2013). Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of Acul in E coli, YhdH is used (see.e.g., Structure of Escherichia coli YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004). This the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and 1pd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase.

In another example of a propionate gene cassette comprises the genes of the Sleeping Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). Recently, this pathway has been considered and utilized for the high yield industrial production of propionate from glycerol (Akawi et al., Engineering Escherichia coli for high-level production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057-1072, the contents of which is herein incorporated by reference in its entirety). In addition, as described herein, it has been found that this pathway is also suitable for production of proprionate from glucose, e.g. by the genetically engineered bacteria of the disclosure. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm (methylmalonyl-CoA mutase) converts succinyl CoA to L-methylmalonylCoA, YgfD is a Sbm-interacting protein kinase with GTPase activity, ygfG (methylmalonylCoA decarboxylase) converts L-methylmalonylCoA into PropionylCoA, and ygfH (propionyl-CoA/succinylCoA transferase) converts propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli; Froese 2009). This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD 18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA.

The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate. One or more of the propionate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

An “acetate gene cassette” or “acetate operon” refers to a set of genes capable of producing acetate in a biosynthetic pathway. Bacteria “synthesize acetate from a number of carbon and energy sources,” including a variety of substrates such as cellulose, lignin, and inorganic gases, and utilize different biosynthetic mechanisms and genes, which are known in the art (Ragsdale et al., 2008). The genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria. Escherichia coli are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994). Several bacteria, such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or CO₂+H₂ into acetate, e.g., using the Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012). Genes in the Wood-Ljungdahl pathway for various bacterial species are known in the art. The acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate. One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

Each gene or gene cassette may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions.

Each gene or gene cassette may be operably linked to a promoter that is induced under low-oxygen conditions. “Operably linked” refers a nucleic acid sequence, e.g., a gene or gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

A “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene or a gene cassette encoding a biosynthetic pathway for producing a metabolic and/or satiety effector molecule, e.g. propionate, and/or immune modulator. In the presence of an inducer of said regulatory region, a metabolic and/or satiety effector and/or immune modulator molecule is expressed. An “indirectly inducible promoter” refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a gene encoding a first molecule, e.g., a transcription factor, which is capable of regulating a second regulatory region that is operably linked to a gene or a gene cassette encoding a biosynthetic pathway for producing a metabolic and/or satiety effector molecule, e.g. propionate, and/or immune modulator. In the presence of an inducer of the first regulatory region, the second regulatory region may be activated or repressed, thereby activating or repressing production of propionate. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.”

“Exogenous environmental condition(s)” or “environmental conditions” refer to settings or circumstances under which the promoter described herein is directly or indirectly induced. The phrase is meant to refer to the environmental conditions external to the engineered microorganism, but endogenous or native to the host subject environment. Thus, “exogenous” and “endogenous” may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease-state, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the disclosure comprises an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics.

As used herein, “exogenous environmental conditions” or “environmental conditions” also refers to settings or circumstances or environmental conditions external to the engineered microorganism, which relate to in vitro culture conditions of the microorganism. “Exogenous environmental conditions” may also refer to the conditions during in vitro growth, production, and manufacture of the organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, low oxygen culture conditions and other conditions under set oxygen concentrations. Such conditions also include the presence of a chemical and/or nutritional inducer, such as tetracycline, arabinose, IPTG, rhamnose, and the like in the culture medium. Such conditions also include the temperatures at which the microorganisms are grown prior to in vivo administration. For example, using certain promoter systems, certain temperatures are permissive to expression of a payload, while other temperatures are non-permissive. Oxygen levels, temperature and media composition influence such exogenous environmental conditions. Such conditions affect proliferation rate, rate of induction of the protein of interest and overall viability and metabolic activity of the strain during strain production.

In some embodiments, the gene or gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule is operably linked to an oxygen level-dependent regulatory region such that the effector molecule is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, the oxygen level-dependent regulatory region is operably linked to a propionate gene cassette; in low oxygen conditions, the oxygen level-dependent regulatory region is activated by a corresponding oxygen level-sensing transcription factor, thereby driving expression of the propionate gene cassette. Examples of oxygen level-dependent transcription factors and corresponding promoters and/or regulatory regions include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1.

TABLE 1 Examples of transcription factors and responsive genes and regulatory regions Transcription Examples of responsive genes, Factor promoters, and/or regulatory regions: FNR nirB, ydfZ, pdhR, focA, ndH, hlyE, narK, narX, narG, yfiD, tdcD Table 4 ANR arcDABC DNR norb, norC

As used herein, a “non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments, “non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In some embodiments, the genetically engineered bacteria of the invention comprise a gene cassette that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene cassette in nature, e.g., a FNR-responsive promoter operably linked to a propionate gene cassette.

“Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive Escherichia coli σ ^(S) promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli σ ³² promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ ⁷⁰ promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis σ ^(A) promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), P_(liaG) (BBa_K823000), P_(lepA) (BBa_K823002), Pveg (BBa_K823003)), a constitutive Bacillus subtilis σ ^(B) promoter (e.g., promoter ctc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_K112706), Pspv from Salmonella (BBa_K112707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)).

“Gut” refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.

“Microorganism” refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, and protozoa. In some aspects, the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules. In certain aspects, the microorganism is engineered to import and/or catabolize certain toxic metabolites, substrates, or other compounds from its environment, e.g., the gut. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites, molecules, or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus.

“Non-pathogenic bacteria” refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria are commensal bacteria, which are present in the indigenous microbiota of the gut. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Pat. No. 6,835,376; U.S. Pat. No. 6,203,797; U.S. Pat. No. 5,589,168; U.S. Pat. No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.

“Probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Pat. No. 5,589,168; U.S. Pat. No. 6,203,797; U.S. Pat. No. 6,835,376). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.

As used herein, the terms “secretion system” or “secretion protein” refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule, e.g., polypeptide from the microbial, e.g., bacterial cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g.,HlyBD. Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the polypeptide to be secreted include a “secretion tag” of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the “passenger” peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the antinflammatory or barrier enhancer molecule(s) into the extracellular milieu. In some embodiments, the secretion system involves the generation of a “leaky” or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nlpl, are deactivated. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes, e.g., selected from 1pp, ompA, ompA, ompF, tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

As used herein, the term “modulate” and its cognates means to alter, regulate, or adjust positively or negatively a molecular or physiological readout, outcome, or process, to effect a change in said readout, outcome, or process as compared to a normal, average, wild-type, or baseline measurement. Thus, for example, “modulate” or “modulation” includes up-regulation and down-regulation. A non-limiting example of modulating a readout, outcome, or process is effecting a change or alteration in the normal or baseline functioning, activity, expression, or secretion of a biomolecule (e.g. a protein, enzyme, cytokine, growth factor, hormone, metabolite, short chain fatty acid, or other compound). Another non-limiting example of modulating a readout, outcome, or process is effecting a change in the amount or level of a biomolecule of interest, e.g. in the serum and/or the gut lumen. In another non-limiting example, modulating a readout, outcome, or process relates to a phenotypic change or alteration in one or more disease symptoms. Thus, “modulate” is used to refer to an increase, decrease, masking, altering, overriding or restoring the normal functioning, activity, or levels of a readout, outcome or process (e.g, biomolecule of interest, and/or molecular or physiological process, and/or a phenotypic change in one or more disease symptoms).

As used herein, “stably maintained” or “stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a propionate gene cassette, which is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and/or propagated. The stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable bacterium may be a genetically modified bacterium comprising a propionate gene cassette, in which the plasmid or chromosome carrying the propionate gene cassette is stably maintained in the host cell, such that the gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro and/or in vivo.

As used herein, the term “treat” and its cognates refer to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, “treat” refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, “treat” refers to inhibiting the progression of a disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, “treat” refers to slowing the progression or reversing the progression of a disease or disorder. As used herein, “prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease or disorder.

Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk of having, or who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Treating metabolic diseases may encompass reducing or eliminating associated symptoms, e.g., weight gain, and does not necessarily encompass the elimination of the underlying disease or disorder, e.g., congenital leptin deficiency. Treating the diseases described herein may encompass increasing levels of propionate, increasing levels of butyrate, and increasing GLP-1, and/or modulating levels of tryptophan and/or its metabolites (e.g., kynurenine), and does not necessarily encompass the elimination of the underlying disease.

As used herein a “pharmaceutical composition” refers to a preparation of genetically engineered bacteria of the invention with other components such as a physiologically suitable carrier and/or excipient.

The phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.

The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

The terms “therapeutically effective dose” and “therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g., obesity. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a metabolic disease. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.

The phrase “and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase “and/or” may be used interchangeably with “at least one of” or “one or more of” the elements in a list.

Bacteria

The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a non-native metabolic and/or satiety effector and/or immune modulator molecule, wherein the gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is controlled by exogenous environmental condition(s). In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.

In some embodiments, the genetically engineered bacteria are Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-positive bacterium of the Enterobacteriaceae family that “has evolved into one of the best characterized probiotics” (Ukena et al., 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors (e.g., E. coli α-hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and is not uropathogenic (Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E. coli Nissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle's “therapeutic efficacy and safety have convincingly been proven” (Ukena et al., 2007). In a recent study in non-human primates, Nissle was well tolerated by female cynomolgus monkeys after 28 days of daily NG dose administration at doses up to 1×1012 CFU/animal. No Nissle related mortality occurred and no Nissle related effects were identified upon clinical observation, body weight, and clinical pathology assessment (see, e.g., PCT/US16/34200).

One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. It is known, for example, that “the clostridial butyrogenic pathway [genes] . . . are widespread in the genome-sequenced clostridia and related species” (Aboulnaga et al., 2013). Furthermore, genes from one or more different species of bacteria can be introduced into one another, e.g., the butyrogenic genes from Peptoclostridium difficile have been expressed in Escherichia coli (Aboulnaga et al., 2013).

Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009). Thus the genetically engineered bacteria may require continued administration. Residence time in vivo may be calculated for the genetically engineered bacteria.

In certain embodiments, the payload(s) described below are expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the payload is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria.

Metabolic Diseases

NASH

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD), where excess fat accumulation in the liver results in chronic inflammation and damage. Nonalcoholic fatty liver disease is a component of metabolic syndrome and a spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Simple liver steatosis is defined as a benign form of NAFLD with minimal risk of progression, in contrast to NASH, which tends to progress to cirrhosis in up to 20% of patients and can subsequently lead to liver failure or hepatocellular carcinoma. NASH affects approximately 3-5% of the population in America, especially in those identified as obese. NASH is characterized by such abnormalities as advanced lipotoxic metabolites, pro-inflammatory substrate, fibrosis, and increased hepatic lipid deposition. If left untreated, NASH can lead to cirrhosis, liver failure, and hepatocellular carcinoma.

Although patients diagnosed with alcoholic steatohepatitis demonstrate similar symptoms and liver damage, NASH develops in individuals who do not consume alcohol, and the underlying causes of NASH are unknown. Hepatic steatosis occurs when the amount of imported and synthesized lipids exceeds the export or catabolism in hepatocytes. An excess intake of fat or carbohydrate is the main cause of hepatic steatosis. NAFLD patients exhibit signs of liver inflammation and increased hepatic lipid accumulation. In addition, the development of NAFLD in obese individuals is closely associated with insulin resistance and other metabolic disorders and thus might be of clinical relevance). Therfore, Possible causative factors include insulin resistance, cytokine imbalance (specifically, an increase in the tumor necrosis factor-alpha (TNF-α)/adiponectin ratio), and oxidative stress resulting from mitochondrial abnormalities.

Currently, there is no accepted approach to treating NASH. Therapy generally involves treating known risk factors such as correction of obesity through diet and exercise, treating hyperglycemia through diet and insulin, avoiding alcohol consumption, and avoiding unnecessary medication. In animal models, administration of butyrate has been shown to reduce hepatic steatosis, inflammation, and fat deposition (see, for example, Jin et al., British J. Nutrition, 114(11):1745-1755, 2015 and Endo et al., PLoS One, 8(5):e63388, 2013). Colonic propionate delivery has also been shown to reduce intrahepatocellular lipid content in NASH patients, including improvements in weight gain and intra-abdominal fat deposition (see, for example, Chambers et al., Gut, gutjnl-2014), and GLP-1 administration has been shown to reduce the degree of lipotoxic metabolites and pro-inflammatory substrates, both of which have been shown to speed NASH development, as well as reduce hepatic lipid deposition (see, for example, Bernsmeier et al., PLoS One, 9(1):e87488, 2014 and Armstrong et al., J. Hepatol., 2015).

The liver has both an arterial and venous blood supply, with the majority of hepatic blood flow coming from the gut via the portal vein. In NASH the liver is exposed to potentially harmful substances derived from the gut (increased perability and reduced intestinal integrity), including translocated bacteria, LPS and endotoxins as well as secreted cytokines. Translocated microbial products might contribute to the pathogenesis of fatty liver disease by several mechanisms, including stimulating pro-inflammatory and profibrotic pathways via a range of cytokines. For example, butyrate and other SCFA, e.g., derived from the microbiota, are known to promote maintaining intestinal integrity.

The role of bile acids in the pathogenesis of NAFLD and NASH has been extensively studied (Leung et al., The Role Of The Gut Microbiota In NAFLD; Nature Reviews I Gastroenterology & Hepatology). For example, in one study study, manipulation of the gut microbiota changed intestinal bile acid composition leading to intestinal antagonism of FRX, the master regulator of bile acid metabolism. This FXR antagonism reduced ceramide synthesis and de novo lipogenesis in the liver (Jiang, C. et al. Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease. J. Clin. Invest. 125, 386-402 (2015)).

Studies have also suggested that rapid weight loss through bariatric surgery (e.g. gastric bypass) is effective in decreasing steatosis, hepatic inflammation, and fibrosis. Other treatments have involved using anti-diabetic medications such as metformin, rosiglitazone, and pioglitazone. Though inconclusive, the studies suggest that the medications stimulate insulin sensitivity in NASH patients, thus alleviating liver damage. In cases were NASH has resulted in advanced cirrhosis, the only treatment is a liver transplant. Regardless, no current treatments are wholly determinative or reliable for treating NASH. Therefore, a need exists for improved therapies and treatments of NASH.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of NAFLD and/or NASH. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate and further comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of NAFLD and/or NASH. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream indole tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more indole tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32.

In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein., e.g., for the treatment, prevention and/or management of NASH

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut e.g., for the treatment, prevention and/or management of NASH. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in

FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, e.g., for the treatment, prevention and/or management of NASH.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios, e.g., for the prevention, management and/or treatment of NASH.

In certain embodiments, one or more of these circuits may be combined for the treatment of NASH and/or NAFLD. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of NASH and/or NAFLD.

Diabetes

Diabetes mellitus type 1 (also known as type 1 diabetes) is a form of diabetes mellitus that results from the autoimmune destruction of the insulin-producing beta cells in the pancreas. The subsequent lack of insulin leads to increased glucose in blood and urine. The classical symptoms are frequent urination, increased thirst, increased hunger, and weight loss. In some embodiments the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of diabetes mellitus.

Diabetes mellitus type 2 is a long term metabolic disorder that is characterized by high blood sugar, insulin resistance, and relative lack of insulin. Common symptoms include increased thirst, frequent urination, and unexplained weight loss. Symptoms may also include increased hunger, feeling tired, and sores that do not heal. Often symptoms come on slowly. Long-term complications from high blood sugar include heart disease, strokes, diabetic retinopathy which can result in blindness, kidney failure, and poor blood flow in the limbs which may lead to amputations.

Insulin resistance (IR) is generally regarded as a pathological condition in which cells fail to respond to the normal actions of the hormone insulin. Normally insulin produced when glucose enters the circulation after a meal triggers glucose uptake into cells. Under conditions of insulin resistance, the cells in the body are resistant to the insulin produced after a meal, preventing glucose uptake and leading to high blood sugar.

The kynurenine hypothesis of diabetes is based on evidence of diabetogenic effects of the kynurenine metabolite Xanthurenic Acid (XA) and the realization that the KP is upregulated by low-grade inflammation and stress, two conditions involved in the pathogenesis of insulin resistance, and of diabetes type I and diabetes type II. Increased concentrations of KYNA and xanthurenic acid (3-Hydroxy KYNA, XA) were detected in the plasma of patients with type 2 diabetes, possibly due to chronic stress or the low-grade inflammation, which are risk factors for T2DM. As such, the production of kynurenine metabolites can function as a regulatory mechanism to attenuate damage by the inflammation-induced production of reactive oxygen species.

Experimental and clinical data have clearly established that besides fat, muscle and liver, pancreatic islet tissue itself is a site of inflammation during obesity and type 2 diabetes. It is therefore conceivable that in parallel to the high free fatty acids and glucose levels, pancreatic islet exposure to increased levels of cytokines may induce dysregulation of islet KP.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of type 2 diabetes. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of type 2 diabetes. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of type 2 diabetes (T2DM). In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein., in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In one embodiment, the genetically engineered bacteria produce IL-22, e.g., for the treatment of diabetes and other metabolic disease described herein.

In certain embodiments, one or more of these circuits may be combined for the treatment of type 2 diabetes. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of type 2 diabetes.

Obesity

Metabolic Syndrome affects approximately 20-30% of the middle-aged population, and represents an increased risk to cardiovascular disorders, the leading cause of death in the United States. Obesity, dyslipidemia, hypertension, and type 2 diabetes are described as metabolic syndrome. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of metabolic syndrome and /or obesity. Several of the metabolites and polypeptides produced by the genetically engineered bacteria are useful for increasing insulin secretion and promoting satiety, e.g. GLP-1.

Obesity is a common, deadly, and costly disease in developed countries which impacts all age groups, race, and gender. Obesity can be classified as an inflammatory disease because it is associated with immune activation and a chronic, low-grade systemic inflammation. Endotoxemia, a process resulting from translocation of endotoxic compounds (lipopolysaccharides [LPS]), of gram-negative intestinal bacteria. In the last decade, it has become evident that insulin resistance and T2DM are characterized by low-grade inflammation. In this respect, LPS trigger a low-grade inflammatory response, and the process of endotoxemia can therefore result in the development of insulin resistance and other metabolic disorders. Several of the metabolites produced by the genetically engineered bacteria described herein are useful in the reduction of inflammation. For example, butyrate, contributes to maintaining intestinal integrity. Other anti-inflammatory metabolites as described herein may also be useful in the treatment of type 2 diaberes.

Over nutrition leads to an excess intake of tryptophan (TRP)—an essential amino acid, a precursor for serotonin (5-HT) and melatonin, and a key player in the caloric intake regulation. Yet, the circulating levels of TRP have been shown to be low in morbidly obese subjects (Brandacher G, Winkler C, Aigner F, et al. Bariatric surgery cannot prevent tryptophan depletion due to chronic immune activation in morbidly obese patients. Obes Surg 2006; 16:541-548).

Serotonin regulates carbohydrate and fat intake (Blundell J E, Lawton C L. Serotonin and dietary fat intake: effects of dexfenfluramine. Metabolism 1995; 44:33-37) , relieves stress which is another caloric intake trigger (Buwalda B, Blom W A, Koolhaas J M, van Dijk G. Behavioral and physiological responses to stress are affected by high-fat feeding in male rats; Physiol Behav 2001; 73:371-377), and inhibits neuropeptide Y (NYP)—one of the most potent orexigenic peptides in the hypothalamus (Jia Y, El-Haddad M, Gendy A, Nguyen T, Ross M G.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1 and/or GLP-1 analog(s). In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of obesity. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including, but not limited, to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, and elsewhere herein, including but not limited to, Tryptamine, Indole-3-acetaldehyde, Indole-3-acetic acid, indole-3- propionic acid, Indole, 6-formylindolo(3,2-b)carbazole, Kynurenic acid, Indole-3-aldehyde; 3,3′ -Diindo lylmethane. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of obesity. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of obesity. Further combinations may include cytokine producing circuits, such as IL-22.

Prader Willi Syndrome

Prader-Willi syndrome (OMIM 176270) is a complex genetic neurodevelopmental disorder with manifested early in failure to thrive, feeding difficulties during infancy, hypogonadism/hypogenitalism, growth hormone deficiency, and typically a paternal 15q11-q13 chromosome deletion. In early childhood trough alduhood, food seeking behaviors and hyperphagia are noted along with a low metabolic rate and decreased physical activity leading to obesity which can be life-threatening, if not controlled. PWS is considered the most common syndromic cause of life threatening obesity in childhood (Buttler et al., Am J Med Genet A. 2015 March; 167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). It has been reported that, when matched for body mass index (BMI), PWS adults had the same prevalence of metabolic syndrome (41.4%) and insulin resistance index as obese controls.

Prader-Willi syndrome (PWS) has no cure. PWS syndrome individuals present with obesity with hyperphagia and deficit of satiety, and in some cases insulin resistance, that persists thoughout youth and adulthood and remains a critical problem in PWS teenagers and adults because it leads to severe complications, such as limb edema, cardiac or respiratory failure, and physical disabilities. Severe obesity, and food seeking therfroe remains the larges problem with PWS. Access to food must be strictly supervised and limited. Therefore, agents which modulate satiety and orh insulin levels may be useful in the treatment of PWS.

In additiona, increased inflammatory markers and cytokine levels in the plasma have been observed in PWS individuals. These cytokines serve as chemoattractants for recruitment of immune cells and indicate an inflammatory component in PWS, which underlies certain aspects of the pathology (Buttler et al., Am J Med Genet A. 2015 March; 167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). Therefore, anti-inflammatory agents may be useful in the treatment of certain aspects of PWS.

In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of PWS. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of PWS. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of PWS.

Metabolic Syndrome

Metabolic syndrome is a clustering of at least three of five of the following medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein (HDL) levels.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of metabolic syndrome. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of metabolic syndrome.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of metabolic syndrome. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of metabolic syndrome.

Cardiovascular Disease

Metabolic syndrome is an important risk factor for cardiovascular disease incidence and mortality, as well as all-cause mortality.

Cardiovascular disease includes coronary artery diseases (CAD) such as angina and myocardial infarction, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, and venous thrombosis. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis, caused inter alia by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol consumption, and the like.

The detection, prevention, and treatment of the underlying risk factors of the metabolic syndrome are a critical approach to lower the cardiovascular disease incidence in the general population.

Cellular adhesion molecules and oxidative stress play a role in the pathogenesis of atherosclerosis in patients with chronic kidney disease (CKD) and uremia. Uremia is condition that occurs when the kidneys no longer filter properly, and is likely to occur s in the final stage of chronic kidney disease. Several studies in CKD patients have shown that tryptophan metabolites along the kynurenine pathway are increased, possibly as consequence of inflammation. Therefore, anti-inflammatory agents may be useful in the treatment of cardiovascular disease, including CKD and artherosclerosis. In some embodiments, the genetically engineered bacteria modulate the levels of one or more of tryptophan, kynurenine, kynurenine downstream metabolites, and other tryptophan metabolites and /or modulate one or more metabolite ratios.

Ischemic stroke, which results from cerebral arterial occlusion, is becoming a major cause of morbidity and mortality in today's society and affects millions of people every year. Currently, the only approved treatment for the acute phase of stroke is the recombinant thrombolytic tissue-type plasminogen activator. Identifying molecules that contribute to the ischemic damage may help to elucidate potential therapeutic targets. In some embodiments the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of ischemia and stroke. Inflammation and oxidative stress are also involved in brain damage following stroke, and tryptophan oxidation along the kynurenine pathway contributes to the modulation of oxidative stress.

In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of cardiovascular disease, including but not limited to, one or more of coronary artery diseases, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, venous thrombosis, ischemic stroke, and/or chronic kidney disease. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of cardiovascular disease, including but not limited to, one or more of coronary artery diseases, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, venous thrombosis,.ischemic stroke, and/or chronic kidney disease.

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of cardiovascular disease.

In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease.

In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

In certain embodiments, one or more of these circuits may be combined for the treatment of cardionvascular disorders. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of cardionvascular disorders.

Metabolic and Satiety Effector Molecules, and Modulators of Inflammation

The genetically engineered bacteria comprise a gene encoding a non-native metabolic and/or satiety effector and/or immune modulator molecule, and/or a gene cassette encoding a biosynthetic pathway capable of producing a metabolic and/or satiety effector and/or immune modulator molecule. In some embodiments, the metabolic and/or satiety effector molecule is selected from the group consisting of n-acyl-phophatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, entero statin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA). A molecule may be primarily a metabolic effector, or primarily a satiety effector. Alternatively, a molecule may be both a metabolic and satiety effector.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) which are capable of producing an effector, which can modulate the inflammatory status. Non-limiting examples include short chain fatty acids, and tryptophan and its metabolites, including indoles, as described herein.

In some embodiments, the genetically engineered bacteria comprise a gene encoding a non-native metabolic and/or satiety effector and/or immune modulator molecule, and/or a gene cassette encoding a biosynthetic pathway capable of producing a metabolic and/or satiety effector and/or immune modulator molecule, and further comprise gene sequence(s) and/or gene cassette(s) which are capable of producing one or more immune modulators or effector molecules which can modulate the inflammatory status, including, for example, short chain fatty acids, and tryptophan and its metabolites, including indoles, as described herein.

The effect of the genetically engineered bacteria on the inflammatory status can be measured by methods known in the art, e.g., plasma can be drawn before and after administraton of the genetically engineered bacteria. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and plasma viscosity (PV) blood tests are commonly used to detect this increase n inflammation. In some embodiments the genetically engineered bacteria modulate, e.g. decrease or increase, levels of inflammatory markers, eg.. C-reactive protein (CRP).

In some embodiments, the genetically engineered bacteria modulate, e.g. decrease, levels of inflammatory growth factors and cytokines, e.g., IL-1(3, IL-6, and/or TNF-α and proinflammatory signaling, e.g. NF-kappaB signaling. In some embodiments the genetically engineered bacteria modulate, e.g. increase, levels of anti-inflammatory growth factors and cytokines, e.g., IL4, IL-10, IL-13, IFN-alpha and/or transforming growth factor-beta.

In some embodiments, the genetically engineered bacteria produce effectors, which bind to and stimulate the aromatic hydrocarbon receptor. In some embodiments the genetically engineered bacteria stimulate AHR signaling in immune cell types, including T cells, B cells, NK cells, macrophages, and dendritic cells (DCs), and/or in epithelial cells. In some embodiments, the genetically engineered bacteria modulate, e.g., increase the levels of IL-22, e.g., through stimulation of AHR.

In some emobodiments, the genetically engineered bacteria may reduce gut permeability. In some embodiments, the genetically engineered bacteria may reduce the amounts of LPS and in the circulation, which are increase in metabolic disease, e.g., in NASH.

The gene or gene cassette for producing the metabolic and/or satiety effector molecule and/or modulator of inflammation may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions, molecules, or metabolites specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.

The gene or gene cassette for producing the metabolic and/or satiety effector and/or modulator of inflammation may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing expression of the metabolic and/or satiety effector and/or immune modulator molecule. In some embodiments, expression from the chromosome may be useful for increasing stability of expression of the metabolic and/or satiety effector molecule. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the propionate biosynthesis gene cassette may be integrated into the bacterial chromosome. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g. FIG. 57). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. In some embodiments, the genetically engineered bacteria of the invention are capable of expressing a metabolic and/or satiety effector and/or immune modulator molecule that is encoded by a single gene, e.g., the molecule is GLP-1 and encoded by the GLP-1 gene.

One of skill in the art would appreciate that additional genes and gene cassettes capable of producing metabolic and/or satiety effector molecules and/or modulator of inflammation are known in the art and may be expressed by the genetically engineered bacteria of the invention. In some embodiments, the gene or gene cassette for producing a therapeutic molecule also comprises additional transcription and translation elements, e.g., a ribosome binding site, to enhance expression of the therapeutic molecule.

In some embodiments, the genetically engineered bacteria produce two or more metabolic and/or satiety effector molecules and/or modulator of inflammation. In certain embodiments, the two or more molecules behave synergistically to ameliorate metabolic disease. In some embodiments, the genetically engineered bacteria express at least one metabolic effector molecule and at least one satiety effector molecule and at least one modulator of inflammation.

Short Chain Fatty Acids

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. In rodent models of genetic or diet-induced obesity, supplementation of butyrate in diet, and oral administration of acetate was shown to suppress weight gain independent of food intake suppression; Propionate was reported to inhibit food intake in humans (see, e.g., Lin et al., Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms, and refernces therein). Therefore, the production of SCFAs is likely efficacious in the treatment of metabolic syndrome and related disorders, and/or diabetes type2, and/or obesity.

SCFAs represent a major constituent of the luminal contents of the colon. Among SCFAs butyrate is believed to play an important role for epithelial homeostasis. Acetate and propionate have anti-inflammatory properties, which are comparable to those of butyrate (Tedelind et al., World J Gastroenterol. 2007 May 28; 13(20): 2826-2832. Acetate and propionate, similar to butyrate, inhibit TNFa-mediated activation of the NF-κB pathway. These findings suggest that propionate and acetate, in addition to butyrate, could be efficacious in the treatment of inflammatory conditions.

Propionate

In alternate embodiments, the genetically engineered bacteria of the invention are capable of producing a metabolic and/or satiety effector molecule, e.g., propionate that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes.

In some embodiments, the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions. The genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 2). Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. In some embodiments, the genetically engineered bacteria of the invention comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In some embodiments, the rate limiting step catalyzed by the Acr enzyme, is replaced by the AcuI from R. sphaeroides, which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of Acul in E coli, yhdH is used. This propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrAf^(fbr), thrB, thrC, ilvA^(fbr), aceE, aceF, and lpd, and optionally further comprise tesB. In another embodiment, the propionate gene cassette comprises the genes of the Sleepting Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm converts succinyl CoA to L-methylmalonylCoA, ygfG converts L-methylmalonylCoA into PropionylCoA, and ygfH converts propionylCoA into propionate and succinate into succinylCoA.

This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD_18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA.

The genes may be codon-optimized, and translational and transcriptional elements may be added. Table 2-4 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette. Table 5 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes.

TABLE 2 Propionate Cassette Sequences (Acrylate Pathway) Gene sequence Description pct ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACT SEQ ID NO: 1 GATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGTGGGTA ACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTC CTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCGG CAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGC ATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGACC GTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGC CTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATA TTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGC ACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGATAT TACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGCAGG AATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATT CGTGGCACCTATGCGGACGAGAGTGGTAACATCACCTTTGAAAA AGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGA AGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTC AAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTAT CTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCATCAAC AGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCAC CGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCGCGAA GAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACG TTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCC GTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTCACAGC GGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCT TTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGGGCTAC CAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTTACCT GGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCC GCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAACATT ACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTACAGC CGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCG TTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAA ATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGT TACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGACG GTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCTGCAGACC CAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCGTGA CGCAAACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAG AAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTAA lcdA ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCATA SEQ ID NO: 2 CTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCC GCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGGAA TTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACTCA TGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGG AAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCCTAC GGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGC CATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCTG CTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATATC TGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACT CGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACCA TGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGTTC CGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTT CGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCGTA GCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATACAAG CCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTT AATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCTTTA AAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATC TACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAGG TATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCTATGA AGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGCCCTT TGGGACCTGCACTATGACGCTAACGACGAATCTATGCACTCTAT GGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGCAGA ACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTG GATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGATGAG TTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACG GTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGC GTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCCTGGT TGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAA lcdB ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGC SEQ ID NO: 3 CGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACAG GTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAGAA ATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGCTT TTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCGAG GGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTG CGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTCCC CAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAGCG GCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCACA ACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGG AAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGACGC AGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCTTA AGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAGATT AAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGT GACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATATCTTTA ATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAA AGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACC GCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGCT CGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGTTAATT AACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTTGCAAT GATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGTAATGT ACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAG GTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCT GCAGTCATTCGTCGAAATGCTTTAA lcdC ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGC SEQ ID NO: 4 GGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTG TCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGAC AAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTA CACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGG ATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTAT TTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCA AGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGC AATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTC CTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAAT GGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCAA GCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTG AGCAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATCT GAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGTT TGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCA GGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTAT CGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCAGCGC TGTATGCTTATGAGGCCGCCCAGAAGAAGTA etfA ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTG SEQ ID NO: 5 GGTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACCGATT TCGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGA AGCAAACTGGTTGGAATTTTGCTGGGGCACGAAGTTGAAGAAAT CGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGTGT GCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGCC AAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGAGGTAAT TTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGCGTT GTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCAC CTGGATATTGATATGAATAAATATGTGGACTTTCTTAGCACCAG TAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAGATA CAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTGATG GCAACGATCATTTGTCCACGCTTCCGTCCCTGTATGAGCACAGT GCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATGG CGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTCGGAT GAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAAACGAA AAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGTTG GTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGCACTGGCT GAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGCGGCTC GCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCATCAGG TTGGACAAACCGGTAAGACCGTGCACCCGAAAGTCTACGTGGCG CTGGGTATTAGTGGGGCTATCCAGCATAAGGCTGGGATGCAAGA CTCTGAACTGATCATTGCCGTCAACAAAGACGAAACGGCGCCTA TCTTCGACTGCGCCGATTATGGCATCACCGGTGATTTATTTAAA ATCGTACCGATGATGATCGACGCGATCAAAGAGGGTAAAAACGC ATGA acrB ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGG SEQ ID NO: 6 CAAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAA TGGCAGCGATTATTAACCCGGACGATATGTCCGCGATCGAACAG GCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGCT TACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATTA TTGCAATGGGGGCCGACGATGGTGTGCTGATTTCGGCCCGTGAA TTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCGC GGCAATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTTT GCGGTCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCCT CAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCGC AGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATGT TGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTCTG ATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGAC TCTCAACGGTATTATGGAATGCTACTCCAAGCCGCTCCTCGTTC TCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGAT ACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTT TACGCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCACCG ATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAAA CATGTCATCTAA acrC ATGTTCTTACTGAAGATTAAAAAAGAACGTATGAAACGCATGGA SEQ ID NO: 7 CTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCGC GTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGATT GATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATGGC GGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGGTG GCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCGTGTCAGAA TTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATTCA TCTTATCGCGCCGCAGGCAATCTACAAATATGGGACCAAAGAAC AGAAAGAGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAACTG GGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATGCCGG CGCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAACGAGT ACGTGCTGAATGGCACCAAATGCTTTATCAGCGGGGGCGGGCGC GCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAAAAAGG TCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGACCCCGG GCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGATCGCAGGT TCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCGTTCCGGC TGCCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAAATTGCTA TGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGCTCAAGCA ATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGT TCACGAGCGCATTCAATTTGGTAAACCGATCGCGAATCTGCAGG GAATTCAATGGTATATCGCGGATATGGCGACCAAAACCGCCGCG GCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAAGACGCGGG TAAACCGTTCACAAAGGAATCTGCTATGTGCAAGCTGAACGCCT CCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATTCAC GGGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGTATGTA TCGCGATGCTAAGATTACGGAAATTTACGAGGGGACATCAGAAA TCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAACGCTAA thr^(fbr) ATGCGAGTGTTGAAGTTCGGCGGTACATCAGTGGCAAATGCAGA SEQ ID NO: 8 ACGTTTTCTGCGTGTTGCCGATATTCTGGAAAGCAATGCCAGGC AGGGGCAGGTGGCCACCGTCCTCTCTGCCCCCGCCAAAATCACC AACCACCTGGTGGCGATGATTGAAAAAACCATTAGCGGCCAGGA TGCTTTACCCAATATCAGCGATGCCGAACGTATTTTTGCCGAAC TTTTGACGGGACTCGCCGCCGCCCAGCCGGGGTTCCCGCTGGCG CAATTGAAAACTTTCGTCGATCAGGAATTTGCCCAAATAAAACA TGTCCTGCATGGCATTAGTTTGTTGGGGCAGTGCCCGGATAGCA TCAACGCTGCGCTGATTTGCCGTGGCGAGAAAATGTCGATCGCC ATTATGGCCGGCGTATTAGAAGCGCGCGGTCACAACGTTACTGT TATCGATCCGGTCGAAAAACTGCTGGCAGTGGGGCATTACCTCG AATCTACCGTCGATATTGCTGAGTCCACCCGCCGTATTGCGGCA AGCCGCATTCCGGCTGATCACATGGTGCTGATGGCAGGTTTCAC CGCCGGTAATGAAAAAGGCGAACTGGTGGTGCTTGGACGCAACG GTTCCGACTACTCTGCTGCGGTGCTGGCTGCCTGTTTACGCGCC GATTGTTGCGAGATTTGGACGGACGTTGACGGGGTCTATACCTG CGACCCGCGTCAGGTGCCCGATGCGAGGTTGTTGAAGTCGATGT CCTACCAGGAAGCGATGGAGCTTTCCTACTTCGGCGCTAAAGTT CTTCACCCCCGCACCATTACCCCCATCGCCCAGTTCCAGATCCC TTGCCTGATTAAAAATACCGGAAATCCTCAAGCACCAGGTACGC TCATTGGTGCCAGCCGTGATGAAGACGAATTACCGGTCAAGGGC ATTTCCAATCTGAATAACATGGCAATGTTCAGCGTTTCTGGTCC GGGGATGAAAGGGATGGTCGGCATGGCGGCGCGCGTCTTTGCAG CGATGTCACGCGCCCGTATTTCCGTGGTGCTGATTACGCAATCA TCTTCCGAATACAGCATCAGTTTCTGCGTTCCACAAAGCGACTG TGTGCGAGCTGAACGGGCAATGCAGGAAGAGTTCTACCTGGAAC TGAAAGAAGGCTTACTGGAGCCGCTGGCAGTGACGGAACGGCTG GCCATTATCTCGGTGGTAGGTGATGGTATGCGCACCTTGCGTGG GATCTCGGCGAAATTCTTTGCCGCACTGGCCCGCGCCAATATCA ACATTGTCGCCATTGCTCAGAGATCTTCTGAACGCTCAATCTCT GTCGTGGTAAATAACGATGATGCGACCACTGGCGTGCGCGTTAC TCATCAGATGCTGTTCAATACCGATCAGGTTATCGAAGTGTTTG TGATTGGCGTCGGTGGCGTTGGCGGTGCGCTGCTGGAGCAACTG AAGCGTCAGCAAAGCTGGCTGAAGAATAAACATATCGACTTACG TGTCTGCGGTGTTGCCAACTCGAAGGCTCTGCTCACCAATGTAC ATGGCCTTAATCTGGAAAACTGGCAGGAAGAACTGGCGCAAGCC AAAGAGCCGTTTAATCTCGGGCGCTTAATTCGCCTCGTGAAAGA ATATCATCTGCTGAACCCGGTCATTGTTGACTGCACTTCCAGCC AGGCAGTGGCGGATCAATATGCCGACTTCCTGCGCGAAGGTTTC CACGTTGTCACGCCGAACAAAAAGGCCAACACCTCGTCGATGGA TTACTACCATCAGTTGCGTTATGCGGCGGAAAAATCGCGGCGTA AATTCCTCTATGACACCAACGTTGGGGCTGGATTACCGGTTATT GAGAACCTGCAAAATCTGCTCAATGCAGGTGATGAATTGATGAA GTTCTCCGGCATTCTTTCTGGTTCGCTTTCTTATATCTTCGGCA AGTTAGACGAAGGCATGAGTTTCTCCGAGGCGACCACGCTGGCG CGGGAAATGGGTTATACCGAACCGGACCCGCGAGATGATCTTTC TGGTATGGATGTGGCGCGTAAACTATTGATTCTCGCTCGTGAAA CGGGACGTGAACTGGAGCTGGCGGATATTGAAATTGAACCTGTG CTGCCCGCAGAGTTTAACGCCGAGGGTGATGTTGCCGCTTTTAT GGCGAATCTGTCACAACTCGACGATCTCTTTGCCGCGCGCGTGG CGAAGGCCCGTGATGAAGGAAAAGTTTTGCGCTATGTTGGCAAT ATTGATGAAGATGGCGTCTGCCGCGTGAAGATTGCCGAAGTGGA TGGTAATGATCCGCTGTTCAAAGTGAAAAATGGCGAAAACGCCC TGGCCTTCTATAGCCACTATTATCAGCCGCTGCCGTTGGTACTG CGCGGATATGGTGCGGGCAATGACGTTACAGCTGCCGGTGTCTT TGCTGATCTGCTACGTACCCTCTCATGGAAGTTAGGAGTCTGA thrB ATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGT SEQ ID NO: 9 CGGGTTTGATGTGCTCGGGGCGGCGGTGACACCTGTTGATGGTG CATTGCTCGGAGATGTAGTCACGGTTGAGGCGGCAGAGACATTC AGTCTCAACAACCTCGGACGCTTTGCCGATAAGCTGCCGTCAGA ACCACGGGAAAATATCGTTTATCAGTGCTGGGAGCGTTTTTGCC AGGAACTGGGTAAGCAAATTCCAGTGGCGATGACCCTGGAAAAG AATATGCCGATCGGTTCGGGCTTAGGCTCCAGTGCCTGTTCGGT GGTCGCGGCGCTGATGGCGATGAATGAACACTGCGGCAAGCCGC TTAATGACACTCGTTTGCTGGCTTTGATGGGCGAGCTGGAAGGC CGTATCTCCGGCAGCATTCATTACGACAACGTGGCACCGTGTTT TCTCGGTGGTATGCAGTTGATGATCGAAGAAAACGACATCATCA GCCAGCAAGTGCCAGGGTTTGATGAGTGGCTGTGGGTGCTGGCG TATCCGGGGATTAAAGTCTCGACGGCAGAAGCCAGGGCTATTTT ACCGGCGCAGTATCGCCGCCAGGATTGCATTGCGCACGGGCGAC ATCTGGCAGGCTTCATTCACGCCTGCTATTCCCGTCAGCCTGAG CTTGCCGCGAAGCTGATGAAAGATGTTATCGCTGAACCCTACCG TGAACGGTTACTGCCAGGCTTCCGGCAGGCGCGGCAGGCGGTCG CGGAAATCGGCGCGGTAGCGAGCGGTATCTCCGGCTCCGGCCCG ACCTTGTTCGCTCTGTGTGACAAGCCGGAAACCGCCCAGCGCGT TGCCGACTGGTTGGGTAAGAACTACCTGCAAAATCAGGAAGGTT TTGTTCATATTTGCCGGCTGGATACGGCGGGCGCACGAGTACTG GAAAACTAA thrC ATGAAACTCTACAATCTGAAAGATCACAACGAGCAGGTCAGCTT SEQ ID NO: 10 TGCGCAAGCCGTAACCCAGGGGTTGGGCAAAAATCAGGGGCTGT TTTTTCCGCACGACCTGCCGGAATTCAGCCTGACTGAAATTGAT GAGATGCTGAAGCTGGATTTTGTCACCCGCAGTGCGAAGATCCT CTCGGCGTTTATTGGTGATGAAATCCCACAGGAAATCCTGGAAG AGCGCGTGCGCGCGGCGTTTGCCTTCCCGGCTCCGGTCGCCAAT GTTGAAAGCGATGTCGGTTGTCTGGAATTGTTCCACGGGCCAAC GCTGGCATTTAAAGATTTCGGCGGTCGCTTTATGGCACAAATGC TGACCCATATTGCGGGTGATAAGCCAGTGACCATTCTGACCGCG ACCTCCGGTGATACCGGAGCGGCAGTGGCTCATGCTTTCTACGG TTTACCGAATGTGAAAGTGGTTATCCTCTATCCACGAGGCAAAA TCAGTCCACTGCAAGAAAAACTGTTCTGTACATTGGGCGGCAAT ATCGAAACTGTTGCCATCGACGGCGATTTCGATGCCTGTCAGGC GCTGGTGAAGCAGGCGTTTGATGATGAAGAACTGAAAGTGGCGC TAGGGTTAAACTCGGCTAACTCGATTAACATCAGCCGTTTGCTG GCGCAGATTTGCTACTACTTTGAAGCTGTTGCGCAGCTGCCGCA GGAGACGCGCAACCAGCTGGTTGTCTCGGTGCCAAGCGGAAACT TCGGCGATTTGACGGCGGGTCTGCTGGCGAAGTCACTCGGTCTG CCGGTGAAACGTTTTATTGCTGCGACCAACGTGAACGATACCGT GCCACGTTTCCTGCACGACGGTCAGTGGTCACCCAAAGCGACTC AGGCGACGTTATCCAACGCGATGGACGTGAGTCAGCCGAACAAC TGGCCGCGTGTGGAAGAGTTGTTCCGCCGCAAAATCTGGCAACT GAAAGAGCTGGGTTATGCAGCCGTGGATGATGAAACCACGCAAC AGACAATGCGTGAGTTAAAAGAACTGGGCTACACTTCGGAGCCG CACGCTGCCGTAGCTTATCGTGCGCTGCGTGATCAGTTGAATCC AGGCGAATATGGCTTGTTCCTCGGCACCGCGCATCCGGCGAAAT TTAAAGAGAGCGTGGAAGCGATTCTCGGTGAAACGTTGGATCTG CCAAAAGAGCTGGCAGAACGTGCTGATTTACCCTTGCTTTCACA TAATCTGCCCGCCGATTTTGCTGCGTTGCGTAAATTGATGATGA ATCATCAGTAA ilvA^(fbr) ATGAGTGAAACATACGTGTCTGAGAAAAGTCCAGGAGTGATGGC SEQ ID NO: 11 TAGCGGAGCGGAGCTGATTCGTGCCGCCGACATTCAAACGGCGC AGGCACGAATTTCCTCCGTCATTGCACCAACTCCATTGCAGTAT TGCCCTCGTCTTTCTGAGGAAACCGGAGCGGAAATCTACCTTAA GCGTGAGGATCTGCAGGATGTTCGTTCCTACAAGATCCGCGGTG CGCTGAACTCTGGAGCGCAGCTCACCCAAGAGCAGCGCGATGCA GGTATCGTTGCCGCATCTGCAGGTAACCATGCCCAGGGCGTGGC CTATGTGTGCAAGTCCTTGGGCGTTCAGGGACGCATCTATGTTC CTGTGCAGACTCCAAAGCAAAAGCGTGACCGCATCATGGTTCAC GGCGGAGAGTTTGTCTCCTTGGTGGTCACTGGCAATAACTTCGA CGAAGCATCGGCTGCAGCGCATGAAGATGCAGAGCGCACCGGCG CAACGCTGATCGAGCCTTTCGATGCTCGCAACACCGTCATCGGT CAGGGCACCGTGGCTGCTGAGATCTTGTCGCAGCTGACTTCCAT GGGCAAGAGTGCAGATCACGTGATGGTTCCAGTCGGCGGTGGCG GACTTCTTGCAGGTGTGGTCAGCTACATGGCTGATATGGCACCT CGCACTGCGATCGTTGGTATCGAACCAGCGGGAGCAGCATCCAT GCAGGCTGCATTGCACAATGGTGGACCAATCACTTTGGAGACTG TTGATCCCTTTGTGGACGGCGCAGCAGTCAAACGTGTCGGAGAT CTCAACTACACCATCGTGGAGAAGAACCAGGGTCGCGTGCACAT GATGAGCGCGACCGAGGGCGCTGTGTGTACTGAGATGCTCGATC TTTACCAAAACGAAGGCATCATCGCGGAGCCTGCTGGCGCGCTG TCTATCGCTGGGTTGAAGGAAATGTCCTTTGCACCTGGTTCTGC AGTGGTGTGCATCATCTCTGGTGGCAACAACGATGTGCTGCGTT ATGCGGAAATCGCTGAGCGCTCCTTGGTGCACCGCGGTTTGAAG CACTACTTCTTGGTGAACTTCCCGCAAAAGCCTGGTCAGTTGCG TCACTTCCTGGAAGATATCCTGGGACCGGATGATGACATCACGC TGTTTGAGTACCTCAAGCGCAACAACCGTGAGACCGGTACTGCG TTGGTGGGTATTCACTTGAGTGAAGCATCAGGATTGGATTCTTT GCTGGAACGTATGGAGGAATCGGCAATTGATTCCCGTCGCCTCG AGCCGGGCACCCCTGAGTACGAATACTTGACCTAA aceE ATGTCAGAACGTTTCCCAAATGACGTGGATCCGATCGAAACTCG SEQ ID NO: 12 CGACTGGCTCCAGGCGATCGAATCGGTCATCCGTGAAGAAGGTG TTGAGCGTGCTCAGTATCTGATCGACCAACTGCTTGCTGAAGCC CGCAAAGGCGGTGTAAACGTAGCCGCAGGCACAGGTATCAGCAA CTACATCAACACCATCCCCGTTGAAGAACAACCGGAGTATCCGG GTAATCTGGAACTGGAACGCCGTATTCGTTCAGCTATCCGCTGG AACGCCATCATGACGGTGCTGCGTGCGTCGAAAAAAGACCTCGA ACTGGGCGGCCATATGGCGTCCTTCCAGTCTTCCGCAACCATTT ATGATGTGTGCTTTAACCACTTCTTCCGTGCACGCAACGAGCAG GATGGCGGCGACCTGGTTTACTTCCAGGGCCACATCTCCCCGGG CGTGTACGCTCGTGCTTTCCTGGAAGGTCGTCTGACTCAGGAGC AGCTGGATAACTTCCGTCAGGAAGTTCACGGCAATGGCCTCTCT TCCTATCCGCACCCGAAACTGATGCCGGAATTCTGGCAGTTCCC GACCGTATCTATGGGTCTGGGTCCGATTGGTGCTATTTACCAGG CTAAATTCCTGAAATATCTGGAACACCGTGGCCTGAAAGATACC TCTAAACAAACCGTTTACGCGTTCCTCGGTGACGGTGAAATGGA CGAACCGGAATCCAAAGGTGCGATCACCATCGCTACCCGTGAAA AACTGGATAACCTGGTCTTCGTTATCAACTGTAACCTGCAGCGT CTTGACGGCCCGGTCACCGGTAACGGCAAGATCATCAACGAACT GGAAGGCATCTTCGAAGGTGCTGGCTGGAACGTGATCAAAGTGA TGTGGGGTAGCCGTTGGGATGAACTGCTGCGTAAGGATACCAGC GGTAAACTGATCCAGCTGATGAACGAAACCGTTGACGGCGACTA CCAGACCTTCAAATCGAAAGATGGTGCGTACGTTCGTGAACACT TCTTCGGTAAATATCCTGAAACCGCAGCACTGGTTGCAGACTGG ACTGACGAGCAGATCTGGGCACTGAACCGTGGTGGTCACGATCC GAAGAAAATCTACGCTGCATTCAAGAAAGCGCAGGAAACCAAAG GCAAAGCGACAGTAATCCTTGCTCATACCATTAAAGGTTACGGC ATGGGCGACGCGGCTGAAGGTAAAAACATCGCGCACCAGGTTAA GAAAATGAACATGGACGGTGTGCGTCATATCCGCGACCGTTTCA ATGTGCCGGTGTCTGATGCAGATATCGAAAAACTGCCGTACATC ACCTTCCCGGAAGGTTCTGAAGAGCATACCTATCTGCACGCTCA GCGTCAGAAACTGCACGGTTATCTGCCAAGCCGTCAGCCGAACT TCACCGAGAAGCTTGAGCTGCCGAGCCTGCAAGACTTCGGCGCG CTGTTGGAAGAGCAGAGCAAAGAGATCTCTACCACTATCGCTTT CGTTCGTGCTCTGAACGTGATGCTGAAGAACAAGTCGATCAAAG ATCGTCTGGTACCGATCATCGCCGACGAAGCGCGTACTTTCGGT ATGGAAGGTCTGTTCCGTCAGATTGGTATTTACAGCCCGAACGG TCAGCAGTACACCCCGCAGGACCGCGAGCAGGTTGCTTACTATA AAGAAGACGAGAAAGGTCAGATTCTGCAGGAAGGGATCAACGAG CTGGGCGCAGGTTGTTCCTGGCTGGCAGCGGCGACCTCTTACAG CACCAACAATCTGCCGATGATCCCGTTCTACATCTATTACTCGA TGTTCGGCTTCCAGCGTATTGGCGATCTGTGCTGGGCGGCTGGC GACCAGCAAGCGCGTGGCTTCCTGATCGGCGGTACTTCCGGTCG TACCACCCTGAACGGCGAAGGTCTGCAGCACGAAGATGGTCACA GCCACATTCAGTCGCTGACTATCCCGAACTGTATCTCTTACGAC CCGGCTTACGCTTACGAAGTTGCTGTCATCATGCATGACGGTCT GGAGCGTATGTACGGTGAAAAACAAGAGAACGTTTACTACTACA TCACTACGCTGAACGAAAACTACCACATGCCGGCAATGCCGGAA GGTGCTGAGGAAGGTATCCGTAAAGGTATCTACAAACTCGAAAC TATTGAAGGTAGCAAAGGTAAAGTTCAGCTGCTCGGCTCCGGTT CTATCCTGCGTCACGTCCGTGAAGCAGCTGAGATCCTGGCGAAA GATTACGGCGTAGGTTCTGACGTTTATAGCGTGACCTCCTTCAC CGAGCTGGCGCGTGATGGTCAGGATTGTGAACGCTGGAACATGC TGCACCCGCTGGAAACTCCGCGCGTTCCGTATATCGCTCAGGTG ATGAACGACGCTCCGGCAGTGGCATCTACCGACTATATGAAACT GTTCGCTGAGCAGGTCCGTACTTACGTACCGGCTGACGACTACC GCGTACTGGGTACTGATGGCTTCGGTCGTTCCGACAGCCGTGAG AACCTGCGTCACCACTTCGAAGTTGATGCTTCTTATGTCGTGGT TGCGGCGCTGGGCGAACTGGCTAAACGTGGCGAAATCGATAAGA AAGTGGTTGCTGACGCAATCGCCAAATTCAACATCGATGCAGAT AAAGTTAACCCGCGTCTGGCGTAA aceF ATGGCTATCGAAATCAAAGTACCGGACATCGGGGCTGATGAAGT SEQ ID NO: 13 TGAAATCACCGAGATCCTGGTCAAAGTGGGCGACAAAGTTGAAG CCGAACAGTCGCTGATCACCGTAGAAGGCGACAAAGCCTCTATG GAAGTTCCGTCTCCGCAGGCGGGTATCGTTAAAGAGATCAAAGT CTCTGTTGGCGATAAAACCCAGACCGGCGCACTGATTATGATTT TCGATTCCGCCGACGGTGCAGCAGACGCTGCACCTGCTCAGGCA GAAGAGAAGAAAGAAGCAGCTCCGGCAGCAGCACCAGCGGCTGC GGCGGCAAAAGACGTTAACGTTCCGGATATCGGCAGCGACGAAG TTGAAGTGACCGAAATCCTGGTGAAAGTTGGCGATAAAGTTGAA GCTGAACAGTCGCTGATCACCGTAGAAGGCGACAAGGCTTCTAT GGAAGTTCCGGCTCCGTTTGCTGGCACCGTGAAAGAGATCAAAG TGAACGTGGGTGACAAAGTGTCTACCGGCTCGCTGATTATGGTC TTCGAAGTCGCGGGTGAAGCAGGCGCGGCAGCTCCGGCCGCTAA ACAGGAAGCAGCTCCGGCAGCGGCCCCTGCACCAGCGGCTGGCG TGAAAGAAGTTAACGTTCCGGATATCGGCGGTGACGAAGTTGAA GTGACTGAAGTGATGGTGAAAGTGGGCGACAAAGTTGCCGCTGA ACAGTCACTGATCACCGTAGAAGGCGACAAAGCTTCTATGGAAG TTCCGGCGCCGTTTGCAGGCGTCGTGAAGGAACTGAAAGTCAAC GTTGGCGATAAAGTGAAAACTGGCTCGCTGATTATGATCTTCGA AGTTGAAGGCGCAGCGCCTGCGGCAGCTCCTGCGAAACAGGAAG CGGCAGCGCCGGCACCGGCAGCAAAAGCTGAAGCCCCGGCAGCA GCACCAGCTGCGAAAGCGGAAGGCAAATCTGAATTTGCTGAAAA CGACGCTTATGTTCACGCGACTCCGCTGATCCGCCGTCTGGCAC GCGAGTTTGGTGTTAACCTTGCGAAAGTGAAGGGCACTGGCCGT AAAGGTCGTATCCTGCGCGAAGACGTTCAGGCTTACGTGAAAGA AGCTATCAAACGTGCAGAAGCAGCTCCGGCAGCGACTGGCGGTG GTATCCCTGGCATGCTGCCGTGGCCGAAGGTGGACTTCAGCAAG TTTGGTGAAATCGAAGAAGTGGAACTGGGCCGCATCCAGAAAAT CTCTGGTGCGAACCTGAGCCGTAACTGGGTAATGATCCCGCATG TTACTCACTTCGACAAAACCGATATCACCGAGTTGGAAGCGTTC CGTAAACAGCAGAACGAAGAAGCGGCGAAACGTAAGCTGGATGT GAAGATCACCCCGGTTGTCTTCATCATGAAAGCCGTTGCTGCAG CTCTTGAGCAGATGCCTCGCTTCAATAGTTCGCTGTCGGAAGAC GGTCAGCGTCTGACCCTGAAGAAATACATCAACATCGGTGTGGC GGTGGATACCCCGAACGGTCTGGTTGTTCCGGTATTCAAAGACG TCAACAAGAAAGGCATCATCGAGCTGTCTCGCGAGCTGATGACT ATTTCTAAGAAAGCGCGTGACGGTAAGCTGACTGCGGGCGAAAT GCAGGGCGGTTGCTTCACCATCTCCAGCATCGGCGGCCTGGGTA CTACCCACTTCGCGCCGATTGTGAACGCGCCGGAAGTGGCTATC CTCGGCGTTTCCAAGTCCGCGATGGAGCCGGTGTGGAATGGTAA AGAGTTCGTGCCGCGTCTGATGCTGCCGATTTCTCTCTCCTTCG ACCACCGCGTGATCGACGGTGCTGATGGTGCCCGTTTCATTACC ATCATTAACAACACGCTGTCTGACATTCGCCGTCTGGTGATGTA A lpd ATGAGTACTGAAATCAAAACTCAGGTCGTGGTACTTGGGGCAGG SEQ ID NO: 14 CCCCGCAGGTTACTCCGCTGCCTTCCGTTGCGCTGATTTAGGTC TGGAAACCGTAATCGTAGAACGTTACAACACCCTTGGCGGTGTT TGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGT AGCAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGTA TCGTCTTCGGCGAACCGAAAACCGATATCGACAAGATTCGTACC TGGAAAGAGAAAGTGATCAATCAGCTGACCGGTGGTCTGGCTGG TATGGCGAAAGGCCGCAAAGTCAAAGTGGTCAACGGTCTGGGTA AATTCACCGGGGCTAACACCCTGGAAGTTGAAGGTGAGAACGGC AAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCGGGTTC TCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCCGCGTA TCTGGGACTCCACTGACGCGCTGGAACTGAAAGAAGTACCAGAA CGCCTGCTGGTAATGGGTGGCGGTATCATCGGTCTGGAAATGGG CACCGTTTACCACGCGCTGGGTTCACAGATTGACGTGGTTGAAA TGTTCGACCAGGTTATCCCGGCAGCTGACAAAGACATCGTTAAA GTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGATGCTGGA AACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGCATTTATG TGACGATGGAAGGCAAAAAAGCACCCGCTGAACCGCAGCGTTAC GACGCCGTGCTGGTAGCGATTGGTCGTGTGCCGAACGGTAAAAA CCTCGACGCAGGCAAAGCAGGCGTGGAAGTTGACGACCGTGGTT TCATCCGCGTTGACAAACAGCTGCGTACCAACGTACCGCACATC TTTGCTATCGGCGATATCGTCGGTCAACCGATGCTGGCACACAA AGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTATCGCCGGTA AGAAACACTACTTCGATCCGAAAGTTATCCCGTCCATCGCCTAT ACCAAACCAGAAGTTGCATGGGTGGGTCTGACTGAGAAAGAAGC GAAAGAGAAAGGCATCAGCTATGAAACCGCCACCTTCCCGTGGG CTGCTTCTGGTCGTGCTATCGCTTCCGACTGCGCAGACGGTATG ACCAAGCTGATTTTCGACAAAGAATCTCACCGTGTGATCGGTGG TGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGGGTGAAATCG GCCTGGCAATCGAAATGGGTTGTGATGCTGAAGACATCGCACTG ACCATCCACGCGCACCCGACTCTGCACGAGTCTGTGGGCCTGGC GGCAGAAGTGTTCGAAGGTAGCATTACCGACCTGCCGAACCCGA AAGCGAAGAAGAAGTAA tesB ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA SEQ ID NO: 15 AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAG GTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTG TATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGA TTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGC GCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTA TATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATC AAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCG GAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGT GCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTC CGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCA CATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGA CCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTA ACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTC GAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGTT CCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGG AGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAG TTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGA AGGGGTGATGCGTAATCACAATTAA acuI ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGT SEQ ID NO: 16 CTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGACG TTTTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGCC CTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGAT GGTACCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCC ACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGG GGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCG CGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGACT TACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGGCGATG TTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGTAA TGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGCTCCG TTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCGGCA GTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTTGGG GGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAGGTAC GCCCGCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGCG GGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGTATCG CGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGATCTGC CCGCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTGGCA GGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGCAGC GTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGGAGG AGATGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGACAGCA CCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATTGTTATCCC CGTAACGCCCTAA

TABLE 3 Propionate Cassette Sequences Sleeping Beauty Operon Sbm ATGTCTAACGTGCAGGAGTGGCAACAGCTTGCCAACAAGGAAT SEQ ID NO: 17 TGAGCCGTCGGGAGAAAACTGTCGACTCGCTGGTTCATCAAAC CGCGGAAGGGATCGCCATCAAGCCGCTGTATACCGAAGCCGAT CTCGATAATCTGGAGGTGACAGGTACCCTTCCTGGTTTGCCGC CCTACGTTCGTGGCCCGCGTGCCACTATGTATACCGCCCAACC GTGGACCATCCGTCAGTATGCTGGTTTTTCAACAGCAAAAGAG TCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAAAG GTCTTTCCGTTGCGTTTGACCTTGCCACCCACCGTGGCTACGA CTCCGATAACCCGCGCGTGGCGGGCGACGTCGGCAAAGCGGGC GTCGCTATCGACACCGTGGAAGATATGAAAGTCCTGTTCGACC AGATCCCGCTGGATAAAATGTCGGTTTCGATGACCATGAATGG CGCAGTGCTACCAGTACTGGCGTTTTATATCGTCGCCGCAGAA GAGCAAGGTGTTACACCTGATAAACTGACCGGCACCATTCAAA ACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATATTTA CCCACCAAAACCGTCAATGCGCATTATCGCCGACATCATCGCC TGGTGTTCCGGCAACATGCCGCGATTTAATACCATCAGTATCA GCGGTTACCACATGGGTGAAGCGGGTGCCAACTGCGTGCAGCA GGTAGCATTTACGCTCGCTGATGGGATTGAGTACATCAAAGCA GCAATCTCTGCCGGACTGAAAATTGATGACTTCGCTCCTCGCC TGTCGTTCTTCTTCGGCATCGGCATGGATCTGTTTATGAACGT CGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGCGGTC AGTGGATTTGGCGCACAGGACCCGAAATCACTGGCGCTGCGTA CCCACTGCCAGACCTCAGGCTGGAGCCTGACTGAACAGGATCC GTATAACAACGTTATCCGCACCACCATTGAAGCGCTGGCTGCG ACGCTGGGCGGTACTCAGTCACTGCATACCAACGCCTTTGACG AAGCGCTTGGTTTGCCTACCGATTTCTCAGCACGCATTGCCCG CAACACCCAGATCATCATCCAGGAAGAATCAGAACTCTGCCGC ACCGTCGATCCACTGGCCGGATCCTATTACATTGAGTCGCTGA CCGATCAAATCGTCAAACAAGCCAGAGCTATTATCCAACAGAT CGACGAAGCCGGTGGCATGGCGAAAGCGATCGAAGCAGGTCTG CCAAAACGAATGATCGAAGAGGCCTCAGCGCGCGAACAGTCGC TGATCGACCAGGGCAAGCGTGTCATCGTTGGTGTCAACAAGTA CAAACTGGATCACGAAGACGAAACCGATGTACTTGAGATCGAC AACGTGATGGTGCGTAACGAGCAAATTGCTTCGCTGGAACGCA TTCGCGCCACCCGTGATGATGCCGCCGTAACCGCCGCGTTGAA CGCCCTGACTCACGCCGCACAGCATAACGAAAACCTGCTGGCT GCCGCTGTTAATGCCGCTCGCGTTCGCGCCACCCTGGGTGAAA TTTCCGATGCGCTGGAAGTCGCTTTCGACCGTTATCTGGTGCC AAGCCAGTGTGTTACCGGCGTGATTGCGCAAAGCTATCATCAG TCTGAGAAATCGGCCTCCGAGTTCGATGCCATTGTTGCGCAAA CGGAGCAGTTCCTTGCCGACAATGGTCGTCGCCCGCGCATTCT GATCGCTAAGATGGGCCAGGATGGACACGATCGCGGCGCGAAA GTGATCGCCAGCGCCTATTCCGATCTCGGTTTCGACGTAGATT TAAGCCCGATGTTCTCTACACCTGAAGAGATCGCCCGCCTGGC CGTAGAAAACGACGTTCACGTAGTGGGCGCATCCTCACTGGCT GCCGGTCATAAAACGCTGATCCCGGAACTGGTCGAAGCGCTGA AAAAATGGGGACGCGAAGATATCTGCGTGGTCGCGGGTGGCGT CATTCCGCCGCAGGATTACGCCTTCCTGCAAGAGCGCGGCGTG GCGGCGATTTATGGTCCAGGTACACCTATGCTCGACAGTGTGC GCGACGTACTGAATCTGATAAGCCAGCATCATGATTAA ygfD ATGATTAATGAAGCCACGCTGGCAGAAAGTATTCGCCGCTTAC SEQ ID NO: 18 GTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCTGGT GGAAAGCCGTCACCCGCGTCATCAGGCACTAAGTACGCAGCTG CTTGATGCCATTATGCCGTACTGCGGTAACACCCTGCGACTGG GCGTTACCGGCACCCCCGGCGCGGGGAAAAGTACCTTTCTTGA GGCCTTTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCGCG GTTATTGCGGTCGATCCCAGCAGCCCGGTCACTGGCGGTAGCA TTCTCGGGGATAAAACCCGCATGAATGACCTGGCGCGTGCCGA AGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCTGGGC GGTGCCAGTCAGCGAGCGCGGGAATTAATGCTGTTATGCGAAG CAGCGGGTTATGACGTAGTGATTGTCGAAACGGTTGGCGTCGG GCAGTCGGAAACAGAAGTCGCCCGCATGGTGGACTGTTTTATC TCGTTGCAAATTGCCGGTGGCGGCGATGATCTGCAGGGCATTA AAAAAGGGCTGATGGAAGTGGCTGATCTGATCGTTATCAACAA AGACGATGGCGATAACCATACCAATGTCGCCATTGCCCGGCAT ATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAATACGACG AATGGCAGCCACGGGTTCTGACTTGTAGCGCACTGGAAAAACG TGGAATCGATGAGATCTGGCACGCCATCATCGACTTCAAAACC GCGCTAACTGCCAGTGGTCGTTTACAACAAGTGCGGCAACAAC AATCGGTGGAATGGCTGCGTAAGCAGACCGAAGAAGAAGTACT GAATCACCTGTTCGCGAATGAAGATTTCGATCGCTATTACCGC CAGACGCTTTTAGCGGTCAAAAACAATACGCTCTCACCGCGCA CCGGCCTGCGGCAGCTCAGTGAATTTATCCAGACGCAATATTT TGATTAA ygfG ATGTCTTATCAGTATGTTAACGTTGTCACTATCAACAAAGTGG SEQ ID NO: 19 CGGTCATTGAGTTTAACTATGGCCGAAAACTTAATGCCTTAAG TAAAGTCTTTATTGATGATCTTATGCAGGCGTTAAGCGATCTC AACCGGCCGGAAATTCGCTGTATCATTTTGCGCGCACCGAGTG GATCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCC GTCTGGCGGTCGCGATCCGCTCTCCTATGATGATCCATTGCGT CAAATCACCCGCATGATCCAAAAATTCCCGAAACCGATCATTT CGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTGAAATGAT CATGAGTTCCGATCTGATCATCGCCGCCAGTACCTCAACCTTC TCAATGACGCCTGTAAACCTCGGCGTCCCGTATAACCTGGTCG GCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAA AGAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCG CTGGCTGTCGGCATCCTCAACCATGTTGTGGAAGTGGAAGAAC TGGAAGATTTCACCTTACAAATGGCGCACCACATCTCTGAGAA AGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGCTGCGTGTA CTGGGCGAAGCACACACCATGAACTCCGATGAATTTGAACGTA TTCAGGGGATGCGCCGCGCGGTGTATGACAGCGAAGATTACCA GGAAGGGATGAACGCTTTCCTCGAAAAACGTAAACCTAATTTC GTTGGTCATTAA ygfH ATGGAAACTCAGTGGACAAGGATGACCGCCAATGAAGCGGCAG SEQ ID NO: 20 AAATTATCCAGCATAACGACATGGTGGCATTTAGCGGCTTTAC CCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCGC AGAGCTAACGAACAGCATGAGGCCAAAAAGCCGTATCAAATTC GCCTTCTGACGGGTGCGTCAATCAGCGCCGCCGCTGACGATGT ACTTTCTGACGCCGATGCTGTTTCCTGGCGTGCGCCATATCAA ACATCGTCCGGTTTACGTAAAAAGATCAATCAGGGCGCGGTGA GTTTCGTTGACCTGCATTTGAGCGAAGTGGCGCAAATGGTCAA TTACGGTTTCTTCGGCGACATTGATGTTGCCGTCATTGAAGCA TCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGCGGGA TCGGTAATGCGCCGACCTGGCTGCTGCGGGCGAAGAAAGTGAT CATTGAACTCAATCACTATCACGATCCGCGCGTTGCAGAACTG GCGGATATTGTGATTCCTGGCGCGCCACCGCGGCGCAATAGCG TGTCGATCTTCCATGCAATGGATCGCGTCGGTACCCGCTATGT GCAAATCGATCCGAAAAAGATTGTCGCCGTCGTGGAAACCAAC TTGCCCGACGCCGGTAATATGCTGGATAAGCAAAATCCCATGT GCCAGCAGATTGCCGATAACGTGGTCACGTTCTTATTGCAGGA AATGGCGCATGGGCGTATTCCGCCGGAATTTCTGCCGCTGCAA AGTGGCGTGGGCAATATCAATAATGCGGTAATGGCGCGTCTGG GGGAAAACCCGGTAATTCCTCCGTTTATGATGTATTCGGAAGT GCTACAGGAATCGGTGGTGCATTTACTGGAAACCGGCAAAATC AGCGGGGCCAGCGCCTCCAGCCTGACAATCTCGGCCGATTCCC TGCGCAAGATTTACGACAATATGGATTACTTTGCCAGCCGCAT TGTGTTGCGTCCGCAGGAGATTTCCAATAACCCGGAAATCATC CGTCGTCTGGGCGTCATCGCTCTGAACGTCGGCCTGGAGTTTG ATATTTACGGGCATGCCAACTCAACACACGTAGCCGGGGTCGA TCTGATGAACGGCATCGGCGGCAGCGGTGATTTTGAACGCAAC GCGTATCTGTCGATCTTTATGGCCCCGTCGATTGCTAAAGAAG GCAAGATCTCAACCGTCGTGCCAATGTGCAGCCATGTTGATCA CAGCGAACACAGCGTCAAAGTGATCATCACCGAACAAGGGATC GCCGATCTGCGCGGTCTTTCCCCGCTTCAACGCGCCCGCACTA TCATTGATAATTGTGCACATCCTATGTATCGGGATTATCTGCA TCGCTATCTGGAAAATGCGCCTGGCGGACATATTCACCACGAT CTTAGCCACGTCTTCGACTTACACCGTAATTTAATTGCAACCG GCTCGATGCTGGGTTAA

TABLE 4 Sequences of Propionate Cassette from Propioni Bacteria Description Sequence mutA ATGAGCAGCACGGATCAGGGGACCAACCCCGCCGACACTGACG SEQ ID NO: 21 ACCTCACTCCCACCACACTCAGTCTGGCCGGGGATTTCCCCAA GGCCACTGAGGAGCAGTGGGAGCGCGAAGTTGAGAAGGTATTC AACCGTGGTCGTCCACCGGAGAAGCAGCTGACCTTCGCCGAGT GTCTGAAGCGCCTGACGGTTCACACCGTCGATGGCATCGACAT CGTGCCGATGTACCGTCCGAAGGACGCGCCGAAGAAGCTGGGT TACCCCGGCGTCACCCCCTTCACCCGCGGCACCACGGTGCGCA ACGGTGACATGGATGCCTGGGACGTGCGCGCCCTGCACGAGGA TCCCGACGAGAAGTTCACCCGCAAGGCGATCCTTGAAGACCTG GAGCGTGGCGTCACCTCCCTGTTGTTGCGCGTTGATCCCGACG CGATCGCACCCGAGCACCTCGACGAGGTCCTCTCCGACGTCCT GCTGGAAATGACCAAGGTGGAGGTCTTCAGCCGCTACGACCAG GGTGCCGCCGCCGAGGCCTTGATGGGCGTCTACGAGCGCTCCG ACAAGCCGGCGAAGGACCTGGCCCTGAACCTGGGCCTGGATCC CATCGGCTTCGCGGCCCTGCAGGGCACCGAGCCGGATCTGACC GTGCTCGGTGACTGGGTGCGCCGCCTGGCGAAGTTCTCACCGG ACTCGCGCGCCGTCACGATCGACGCGAACGTCTACCACAACGC CGGTGCCGGCGACGTGGCAGAGCTCGCTTGGGCACTGGCCACC GGCGCGGAGTACGTGCGCGCCCTGGTCGAACAGGGCTTCAACG CCACAGAGGCCTTCGACACGATCAACTTCCGTGTCACCGCCAC CCACGACCAGTTCCTCACGATCGCCCGTCTTCGCGCCCTGCGC GAGGCATGGGCCCGCATCGGCGAGGTCTTTGGCGTGGACGAGG ACAAGCGCGGCGCTCGCCAGAATGCGATCACCAGTTGGCGTGA GCTCACCCGCGAAGACCCCTATGTCAACATCCTTCGCGGTTCG ATTGCCACCTTCTCCGCCTCCGTTGGCGGGGCCGAGTCGATCA CGACGCTGCCCTTCACCCAGGCCCTCGGCCTGCCGGAGGACGA CTTCCCGCTGCGCATCGCGCGCAACACGGGCATCGTGCTCGCC GAAGAGGTGAACATCGGCCGCGTCAACGACCCGGCCGGTGGCT CCTACTACGTCGAGTCGCTCACTCGCACCCTGGCCGACGCTGC CTGGAAGGAATTCCAGGAGGTCGAGAAGCTCGGTGGCATGTCG AAGGCGGTCATGACCGAGCACGTCACCAAGGTGCTCGACGCCT GCAATGCCGAGCGCGCCAAGCGCCTGGCCAACCGCAAGCAGCC GATCACCGCGGTCAGCGAGTTCCCGATGATCGGGGCCCGCAGC ATCGAGACCAAGCCGTTCCCAACCGCTCCGGCGCGCAAGGGCC TGGCCTGGCATCGCGATTCCGAGGTGTTCGAGCAGCTGATGGA TCGCTCCACCAGCGTCTCCGAGCGCCCCAAGGTGTTCCTTGCC TGCCTGGGCACCCGTCGCGACTTCGGTGGCCGCGAGGGCTTCT CCAGCCCGGTATGGCACATCGCCGGTATCGACACCCCGCAGGT CGAAGGCGGCACCACCGCCGAGATCGTCGAGGCGTTCAAGAAG TCGGGCGCCCAGGTGGCCGATCTCTGCTCGTCCGCCAAGATCT ACGCGCAGCAGGGACTTGAGGTTGCCAAGGCGCTCAAGGCCGC CGGCGCGAAGGCCCTGTATCTGTCGGGCGCCTTCAAGGAGTTC GGCGATGACGCCGCCGAGGCCGAGAAGCTGATCGACGGACGCC TGTACATGGGCATGGATGTCGTCGACACCCTGTCCTCCACCCT TGATATCTTGGGAGTCGCGAAGTGA mutB GTGAGCACTCTGCCCCGTTTTGATTCAGTTGACCTGGGCAATG SEQ ID NO: 22 CCCCGGTTCCTGCTGATGCCGCACAGCGCTTCGAGGAGTTGGC CGCCAAGGCCGGCACCGAAGAGGCGTGGGAGACGGCTGAGCAG ATTCCGGTTGGCACCCTGTTCAACGAAGACGTCTACAAGGACA TGGACTGGCTGGACACCTACGCCGGTATCCCGCCGTTCGTCCA CGGCCCATATGCAACCATGTACGCGTTCCGTCCCTGGACGATT CGCCAGTACGCCGGCTTCTCCACGGCCAAGGAGTCCAACGCCT TCTACCGCCGCAACCTTGCGGCGGGCCAGAAGGGCCTGTCGGT TGCCTTCGACCTGCCCACCCACCGCGGCTACGACTCGGACAAT CCCCGCGTCGCCGGTGACGTCGGCATGGCCGGGGTGGCCATCG ACTCCATCTATGACATGCGCGAGCTGTTCGCCGGCATTCCGCT GGACCAGATGAGCGTGTCGATGACCATGAACGGCGCCGTGCTG CCGATCCTGGCCCTCTATGTGGTGACCGCCGAGGAGCAGGGCG TCAAGCCCGAGCAGCTCGCCGGGACGATCCAGAACGACATCCT CAAGGAGTTCATGGTTCGTAACACCTATATCTACCCGCCGCAG CCGAGTATGCGAATCATCTCCGAGATCTTCGCCTACACGAGTG CCAATATGCCGAAGTGGAATTCGATTTCCATTTCCGGCTACCA CATGCAGGAAGCCGGCGCCACGGCCGACATCGAGATGGCCTAC ACCCTGGCCGACGGTGTCGACTACATCCGCGCCGGCGAGTCGG TGGGCCTCAATGTCGACCAGTTCGCGCCGCGTCTGTCCTTCTT CTGGGGCATCGGCATGAACTTCTTCATGGAGGTTGCCAAGCTG CGTGCCGCACGTATGTTGTGGGCCAAGCTGGTGCATCAGTTCG GGCCGAAGAATCCGAAGTCGATGAGCCTGCGCACCCACTCGCA GACCTCCGGTTGGTCGCTGACCGCCCAGGACGTCTACAACAAC GTCGTGCGTACCTGCATCGAGGCCATGGCCGCCACCCAGGGCC ATACCCAGTCGCTGCACACGAACTCGCTCGACGAGGCCATTGC CCTACCGACCGATTTCAGCGCCCGCATCGCCCGTAACACCCAG CTGTTCCTGCAGCAGGAATCGGGCACGACGCGCGTGATCGACC CGTGGAGCGGCTCGGCATACGTCGAGGAGCTCACCTGGGACCT GGCCCGCAAGGCATGGGGCCACATCCAGGAGGTCGAGAAGGTC GGCGGCATGGCCAAGGCCATCGAAAAGGGCATCCCCAAGATGC GCATTGAGGAAGCCGCCGCCCGCACCCAGGCACGCATCGACTC CGGCCGTCAGCCGCTGATCGGCGTGAACAAGTACCGCCTGGAG CACGAGCCGCCGCTCGATGTGCTCAAGGTTGACAACTCCACGG TGCTCGCCGAGCAGAAGGCCAAGCTGGTCAAGCTGCGCGCCGA GCGCGATCCCGAGAAGGTCAAGGCCGCCCTCGACAAGATCACC TGGGCTGCCGCCAACCCCGACGACAAGGATCCGGATCGCAACC TGCTGAAGCTGTGCATCGACGCTGGCCGCGCCATGGCGACGGT CGGCGAGATGAGCGACGCGCTCGAGAAGGTCTTCGGACGCTAC ACCGCCCAGATTCGCACCATCTCCGGTGTGTACTCGAAGGAAG TGAAGAACACGCCTGAGGTTGAGGAAGCACGCGAGCTCGTTGA GGAATTCGAGCAGGCCGAGGGCCGTCGTCCTCGCATCCTGCTG GCCAAGATGGGCCAGGACGGTCACGACCGTGGCCAGAAGGTCA TCGCCACCGCCTATGCCGACCTCGGTTTCGACGTCGACGTGGG CCCGCTGTTCCAGACCCCGGAGGAGACCGCACGTCAGGCCGTC GAGGCCGATGTGCACGTGGTGGGCGTTTCGTCGCTCGCCGGCG GGCATCTGACGCTGGTTCCGGCCCTGCGCAAGGAGCTGGACAA GCTCGGACGTCCCGACATCCTCATCACCGTGGGCGGCGTGATC CCTGAGCAGGACTTCGACGAGCTGCGTAAGGACGGCGCCGTGG AGATCTACACCCCCGGCACCGTCATTCCGGAGTCGGCGATCTC GCTGGTCAAGAAACTGCGGGCTTCGCTCGATGCCTAG GI:18042134 ATGAGTAATGAGGATCTTTTCATCTGTATCGATCACGTGGCAT SEQ ID NO: 23 ATGCGTGCCCCGACGCCGACGAGGCTTCCAAGTACTACCAGGA GACCTTCGGCTGGCATGAGCTCCACCGCGAGGAGAACCCGGAG CAGGGAGTCGTCGAGATCATGATGGCCCCGGCTGCGAAGCTGA CCGAGCACATGACCCAGGTTCAGGTCATGGCCCCGCTCAACGA CGAGTCGACCGTTGCCAAGTGGCTTGCCAAGCACAATGGTCGC GCCGGACTGCACCACATGGCATGGCGTGTCGATGACATCGACG CCGTCAGCGCCACCCTGCGCGAGCGCGGCGTGCAGCTGCTGTA TGACGAGCCCAAGCTCGGCACCGGCGGCAACCGCATCAACTTC ATGCATCCCAAGTCGGGCAAGGGCGTGCTCATCGAGCTCACCC AGTACCCGAAGAACTGA mmdA ATGGCTGAAAACAACAATTTGAAGCTCGCCAGCACCATGGAAG SEQ ID NO: 24 GTCGCGTGGAGCAGCTCGCAGAGCAGCGCCAGGTGATCGAAGC CGGTGGCGGCGAACGTCGCGTCGAGAAGCAACATTCCCAGGGT AAGCAGACCGCTCGTGAGCGCCTGAACAACCTGCTCGATCCCC ATTCGTTCGACGAGGTCGGCGCTTTCCGCAAGCACCGCACCAC GTTGTTCGGCATGGACAAGGCCGTCGTCCCGGCAGATGGCGTG GTCACCGGCCGTGGCACCATCCTTGGTCGTCCCGTGCACGCCG CGTCCCAGGACTTCACGGTCATGGGTGGTTCGGCTGGCGAGAC GCAGTCCACGAAGGTCGTCGAGACGATGGAACAGGCGCTGCTC ACCGGCACGCCCTTCCTGTTCTTCTACGATTCGGGCGGCGCCC GGATCCAGGAGGGCATCGACTCGCTGAGCGGTTACGGCAAGAT GTTCTTCGCCAACGTGAAGCTGTCGGGCGTCGTGCCGCAGATC GCCATCATTGCCGGCCCCTGTGCCGGTGGCGCCTCGTATTCGC CGGCACTGACTGACTTCATCATCATGACCAAGAAGGCCCATAT GTTCATCACGGGCCCCCAGGTCATCAAGTCGGTCACCGGCGAG GATGTCACCGCTGACGAACTCGGTGGCGCTGAGGCCCATATGG CCATCTCGGGCAATATCCACTTCGTGGCCGAGGACGACGACGC CGCGGAGCTCATTGCCAAGAAGCTGCTGAGCTTCCTTCCGCAG AACAACACTGAGGAAGCATCCTTCGTCAACCCGAACAATGACG TCAGCCCCAATACCGAGCTGCGCGACATCGTTCCGATTGACGG CAAGAAGGGCTATGACGTGCGCGATGTCATTGCCAAGATCGTC GACTGGGGTGACTACCTCGAGGTCAAGGCCGGCTATGCCACCA ACCTCGTGACCGCCTTCGCCCGGGTCAATGGTCGTTCGGTGGG CATCGTGGCCAATCAGCCGTCGGTGATGTCGGGTTGCCTCGAC ATCAACGCCTCTGACAAGGCCGCCGAATTCGTGAATTTCTGCG ATTCGTTCAACATCCCGCTGGTGCAGCTGGTCGACGTGCCGGG CTTCCTGCCCGGCGTGCAGCAGGAGTACGGCGGCATCATTCGC CATGGCGCGAAGATGCTGTACGCCTACTCCGAGGCCACCGTGC CGAAGATCACCGTGGTGCTCCGCAAGGCCTACGGCGGCTCCTA CCTGGCCATGTGCAACCGTGACCTTGGTGCCGACGCCGTGTAC GCCTGGCCCAGCGCCGAGATTGCGGTGATGGGCGCCGAGGGTG CGGCAAATGTGATCTTCCGCAAGGAGATCAAGGCTGCCGACGA TCCCGACGCCATGCGCGCCGAGAAGATCGAGGAGTACCAGAAC GCGTTCAACACGCCGTACGTGGCCGCCGCCCGCGGTCAGGTCG ACGACGTGATTGACCCGGCTGATACCCGTCGAAAGATTGCTTC CGCCCTGGAGATGTACGCCACCAAGCGTCAGACCCGCCCGGCG AAGAAGCATGGAAACTTCCCCTGCTGA PFREUD_18870 ATGAGTCCGCGAGAAATTGAGGTTTCCGAGCCGCGCGAGGTTG SEQ ID NO: 25 GTATCACCGAGCTCGTGCTGCGCGATGCCCATCAGAGCCTGAT GGCCACACGAATGGCAATGGAAGACATGGTCGGCGCCTGTGCA GACATTGATGCTGCCGGGTACTGGTCAGTGGAGTGTTGGGGTG GTGCCACGTATGACTCGTGTATCCGCTTCCTCAACGAGGATCC TTGGGAGCGTCTGCGCACGTTCCGCAAGCTGATGCCCAACAGC CGTCTCCAGATGCTGCTGCGTGGCCAGAACCTGCTGGGTTACC GCCACTACAACGACGAGGTCGTCGATCGCTTCGTCGACAAGTC CGCTGAGAACGGCATGGACGTGTTCCGTGTCTTCGACGCCATG AATGATCCCCGCAACATGGCGCACGCCATGGCTGCCGTCAAGA AGGCCGGCAAGCACGCGCAGGGCACCATTTGCTACACGATCAG CCCGGTCCACACCGTTGAGGGCTATGTCAAGCTTGCTGGTCAG CTGCTCGACATGGGTGCTGATTCCATCGCCCTGAAGGACATGG CCGCCCTGCTCAAGCCGCAGCCGGCCTACGACATCATCAAGGC CATCAAGGACACCTACGGCCAGAAGACGCAGATCAACCTGCAC TGCCACTCCACCACGGGTGTCACCGAGGTCTCCCTCATGAAGG CCATCGAGGCCGGCGTCGACGTCGTCGACACCGCCATCTCGTC CATGTCGCTCGGCCCGGGCCACAACCCCACCGAGTCGGTTGCC GAGATGCTCGAGGGCACCGGGTACACCACCAACCTTGACTACG ATCGCCTGCACAAGATCCGCGATCACTTCAAGGCCATCCGCCC GAAGTACAAGAAGTTCGAGTCGAAGACGCTTGTCGACACCTCG ATCTTCAAGTCGCAGATCCCCGGCGGCATGCTCTCCAACATGG AGTCGCAGCTGCGCGCCCAGGGCGCCGAGGACAAGATGGACGA GGTCATGGCAGAGGTGCCGCGCGTCCGCAAGGCCGCCGGCTTC CCGCCCCTGGTCACCCCGTCCAGCCAGATCGTCGGCACGCAGG CCGTGTTCAACGTGATGATGGGCGAGTACAAGAGGATGACCGG CGAGTTCGCCGACATCATGCTCGGCTACTACGGCGCCAGCCCG GCCGATCGCGATCCGAAGGTGGTCAAGTTGGCCGAGGAGCAGT CCGGCAAGAAGCCGATCACCCAGCGCCCGGCCGATCTGCTGCC CCCCGAGTGGGAGGAGCAGTCCAAGGAGGCCGCGGCCCTCAAG GGCTTCAACGGCACCGACGAGGACGTGCTCACCTATGCACTGT TCCCGCAGGTCGCTCCGGTCTTCTTCGAGCATCGCGCCGAGGG CCCGCACAGCGTGGCTCTCACCGATGCCCAGCTGAAGGCCGAG GCCGAGGGCGACGAGAAGTCGCTCGCCGTGGCCGGTCCCGTCA CCTACAACGTGAACGTGGGCGGAACCGTCCGCGAAGTCACCGT TCAGCAGGCGTGA Bccp ATGAAACTGAAGGTAACAGTCAACGGCACTGCGTATGACGTTG SEQ ID NO: 26 ACGTTGACGTCGACAAGTCACACGAAAACCCGATGGGCACCAT CCTGTTCGGCGGCGGCACCGGCGGCGCGCCGGCACCGCGCGCA GCAGGTGGCGCAGGCGCCGGTAAGGCCGGAGAGGGCGAGATTC CCGCTCCGCTGGCCGGCACCGTCTCCAAGATCCTCGTGAAGGA GGGTGACACGGTCAAGGCTGGTCAGACCGTGCTCGTTCTCGAG GCCATGAAGATGGAGACCGAGATCAACGCTCCCACCGACGGCA AGGTCGAGAAGGTCCTTGTCAAGGAGCGTGACGCCGTGCAGGG CGGTCAGGGTCTCATCAAGATCGGCTGA

In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 26) or a functional fragment thereof.

Table 5 lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cattette(s) of the genetically engineered bacteria.

TABLE 5 Polypeptide Sequences for Propionate Synthesis Pct MRKVPIITADEAAKLIKDGDTVTTSGFVGNAIPEALDRAVEKRFLET SEQ ID GEPKNITYVYCGSQGNRDGRGAEHFAHEGLLKRYIAGHWATVPALGK NO: 27 MAMENKMEAYNVSQGALCHLFRDTASHKPGVFTKVGIGTFIDPRNGG GKVNDITKEDIVELVEIKGQEYLFYPAFPIHVALIRGTYADESGNIT FEKEVAPLEGTSVCQAVKNSGGIVVVQVERVVKAGTLDPRHVKVPGI YVDYVVVADPEDHQQSLDCEYDPALSGEHRRPEVVGEPLPLSAKKVI GRRGAIELEKDVAVNLGVGAPEYVASVADEEGIVDFMTLTAESGAIG GVPAGGVRFGASYMADALIDQGYQFDYYDGGGLDLCYLGLAECDEKG NINVSRFGPRIAGCGGFINITQNTPKVFFCGTFTAGGLKVKIEDGKV IIVQEGKQKKFLKAVEQITFMGDVALANKQQVTYITERCVFLLKEDG LHLSEIAPGIDLQTQILDVMDFAPIIDRDANGQIKLMDAALFAEGLM GLKEMKS* lcdA MSLTQGMKAKQLLAYFQGKADQDAREAKARGELVCWSASVAPPEFCV SEQ ID TMGIAMIYPETHAAGIGARKGAMDMLEVADRKGYNVDCCSYGRVNMG NO: 28 YMECLKEAAITGVKPEVLVNSPAADVPLPDLVITCNNICNTLLKWYE NLAAELDIPCIVIDVPFNHTMPIPEYAKAYIADQFRNAISQLEVICG RPFDWKKFKEVKDQTQRSVYHWNRIAEMAKYKPSPLNGFDLFNYMAL IVACRSLDYAEITFKAFADELEENLKAGIYAFKGAEKTRFQWEGIAV WPHLGHTFKSMKNLNSIMTGTAYPALWDLHYDANDESMHSMAEAYTR IYINTCLQNKVEVLLGIMEKGQVDGTVYHLNRSCKLMSFLNVETAEI IKEKNGLPYVSIDGDQTDPRVFSPAQFDTRVQALVEMMEANMAAAE* lcdB MSRVEAILSQLKDVAANPKKAMDDYKAETGKGAVGIMPIYSPEEMVH SEQ ID AAGYLPMGIWGAQGKTISKARTYLPAFACSVMQQVMELQCEGAYDDL NO: 29 SAVIFSVPCDTLKCLSQKWKGTSPVIVFTHPQNRGLEAANQFLVTEY ELVKAQLESVLGVKISNAALENSIAIYNENRAVMREFVKVAADYPQV IDAVSRHAVFKARQFMLKEKHTALVKELIAEIKATPVQPWDGKKVVV TGILLEPNELLDIFNEFKIAIVDDDLAQESRQIRVDVLDGEGGPLYR MAKAWQQMYGCSLATDTKKGRGRMLINKTIQTGADAIVVAMMKFCDP EEWDYPVMYREFEEKGVKSLMIEVDQEVSSFEQIKTRLQSFVEML* lcdC MYTLGIDVGSASSKAVILKDGKDIVAAEVVQVGTGSSGPQRALDKAFE SEQ ID VSGLKKEDISYTVATGYGRFNFSDADKQISEISCHAKGIYFLVPTART NO: 30 IIDIGGQDAKAIRLDDKGGIKQFFMNDKCAAGTGRFLEVMARVLETTL DEMAELDEQATDTAPISSTCTVFAESEVISQLSNGVSRNNIIKGVHLS VASRACGLAYRGGLEKDVVMTGGVAKKAGVVRAVAGVLKTDVIVAPNP   QTTGALGAALYAYEAAQKKX etfA MAFNSAD1NSFRDIWVFCEQREGKLINTDFELISEGRKLADERGSKL SEQ ID VGILLGHEVEEIAKELGGYGADKVIVCDHPELKFYTTDAYAKVLCDV NO: 31 VMEEKPEVILIGATNIGRDLGPRCAARLHTGLTADCTHLDIDHNKYV DFLSTSSTLDISSMTFPMEDTNLKMTRPAFGGHLMATIICPRFRPCM STVRPGVMKKAEFSQEMAQACQVVTRHVNLSDEDLKTKVINIVKETK KIVDLIGAEIIVSVGRGISKDVQGGIALAEKLADAFGNGVVGGSRAV IDSGWLPADHQVGQTGKTVHPKVYVALGISGAIQHKAGMQDSELIIA VNKDETAPIFDCADYGITGDLFKIVPMMIDAIKEGKMA* acrB MRIYVCVKQVPDTSGKVAVHPDGTLNRASMAAIINPDDMSAIEQALK SEQ ID LKDETGCQVTALTMGPPPAEGMLREIIAMGADDGVLISAREFGGSDT NO: 32 FATSQIISAAIHKLGLSNEDMIFCGRQAIDGDTAQVGPQIAEKLSIP QVTYGAGIKKSGDLVLVKRMLEDGYMMIEVETPCLITCIQDKAVKPR YMTLNGIMECYSKPLLVLDYEALKDEPLIELDTIGLKGSPTNIFKSF TPPQKGVGVMLQGTDKEKVEDLVDKLMQKHVI* acrC MFLLKTKKERMKRMDFSLTREQEMLKKLARQFAEIELEPVAEEIDRE SEQ ID HVFPAENFKKMAEIGLTGIGIPKEFGGSGGGTLEKVIAVSEFGKKCM NO: 33 ASASILSIHLIAPQAIYKYGTKEQKETYLPRLTKGGELGAFALTEPN AGSDAGAVKTTAILDSQTNEYVLNGTKCFISGGGRAGVLVIFALTEP KKGLKGMSAIIVEKGTPGFSIGKVESKMGIAGSETAELIFEDCRVPA ANLLGKEGKGFKIAMEALDGARIGVGAQAIGIAEGAIDLSVKYVHER IQFGKPIANLQGIQWYIADMATKTAAARALVEFAAYLEDAGKPFTKE SAMCKLNASENARFVTNLALQIHGGYGYMKDYPLERMYRDAKITEIY EGTSEIHKWAREVMKR* thrAfbr MRVLKFGGTSVANAERFLRVADILESKARQGQVATVLSAPAKITNHL SEQ ID VAMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKIFV NO: 34 DQEFAQIKHVLHGISLLGQCPDSINAALICRGEKMSIAIMAGVLEAR GHMVTVIDPVEKLLAVGHYLESTVDIAESTRRIAASRIFADHMVLMA GFTAGNEKGELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTC DPRQVPDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLI KNTGNPQAPGTLIGASRDEDELPVKGISNLNNMAMFSVSGPGMKGMV GMAARVFAAMSRARISVVLITQSSSEYSISFCVPQSDCVRAERAMQE EFYLELKEGLLEPLAVTERLAIISSSGDGMRTLRGISAKFFAALARA NINIVAIAQRSSERSISSSVNNDDATTGVRVTHQMLFNTDQVIEVFV IGVGGVGGALLEQLKRQQSWLKNKHIDLRVCGVANSKALLTNVHGLN LENWQEELAQAKEPFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQY ADFLREGFHSSTPNKKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGA GLPVIENLQNLLNAGDELMKFSGILSGSLSYIFGKLDEGMSFSEATT LAREMGYTEPDPRDDLSGMDVARKLLILARETGRELELADIEIEPVL PAEFNAEGDVAAFMANLSQLDDLFAARVAKARDEGKVLRYVGNIDED GVCRVKIAEVDGNDPLFKVKNGENALAFYSHYYQPLPLVLRGYGAGN DVTAAGVFADLLRTLSWKLGV* thrB MVKVYAPASSANMSVGFDVLGAAVTPVDGALLGDVVTVEAAETFSLN SEQ ID NLGRFADKLPSEPRENIVYQCWERFCQELGKQIPVAMTLEKNMPIGS NO: 35 GLGSSACSVVAALMAMNEHCGKPLNDTRLLALMGELEGRISGSIHYD NVAPCFLGGMQLMIEENDIISQQVPGFDEWLWVLAYPGIKVSTAEAR AILPAQYRRQDCIAHGRHLAGFIHACYSRQPELAAKLMKDVIAEPYR ERLLPGFRQARQAVAEIGAVASGISGSGPTLFALCDKPETAQRVADW LGKNYLQNQEGFVHICRLDTAGARVLEM* thrC MKLYNLKDHNEQVSFAQAVTQGLGKNQGLFFPHDLPEFSLTEIDEML SEQ ID KLDFVTRSAKILSAFIGDEIPQEILEERVRAAFAFPAPVANVESDVG NO: 36 CLELFHGPTLAFKDFGGRFMAQMLTHIAGDKPVTILTATSGDTGAAV AHAFYGLPNVKVVILYPRGKISPLQEKLFCTLGGNIETVAIDGDFDA CQALVKQAFDDEELKVALGLNSANSINISRLLAQICYYFEAVAQLPQ ETRNQLVVSVPSGNFGDLTAGLLAKSLGLPVKRFIAATNVNDTVPRF LHDGQWSPKATQATLSNAMDVSQPNNWPRVEELFRRKIWQLKELGYA AVDDETTQQTMRELKELGYTSEPHAAVAYRALRDQLNPGEYGLFLGT AHPAKFKESVEAILGETLDLPKELAERADLPLLSHNLPADFAALRKL MMNHQ* ilvA^(fbr) MSETYVSEKSPGVMASGAELIRAADIQTAQARISSVIAPTPLQYCPR SEQ ID LSEETGAEIYLKREDLQDVRSYKIRGALNSGAQLTQEQRDAGIVAAS NO: 37 AGNHAQGVAYVCKSLGVQGRIYVPVQTPKQKRDRIMVHGGEFVSLVV TGNNFDEASAAAHEDAERTGATLIEPFDARNTVIGQGTVAAEILSQL TSMGKSADHVMVPVGGGGLLAGVVSYMADMAPRTAIVGIEPAGAASM QAALRNGGPITLETVDPFVDGAAVKRVGDLNYTIVEKNQGRVHMMSA TEGAVCTEMLDLYQNEGIIAEPAGALSIAGLKEMSFAPGSAVVCIIS GGNNDVLRYAEIAERSLVHRGLKHYFLVNFPQKPGQLRHFLEDILGP DDDITLFEYLKRNNRETGTALVGIHLSEASGLDSLLERMEESAIDSR RLEPGTPEYEYLT* ace MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKG SEQ ID GVNVAAGTG1SNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTV NO: 38 LRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQ GHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFW QFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMD EPESKGAITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGI FEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSK DGAYVREHFFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKK AQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRD RFNVPVSDADIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNF TEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLV PIIADEARTFGMEGLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQ ILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDL CWAAGDQQARGFLIGGTSGRTTLNGEGLQHEDGHSHIQSLTIPNCIS YDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEG AEEGIRKGIYKLETIEGSKGKVQLLGSGSXLRHVREAAEILAKDYGV GSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAV ASTDYMKLFAEQVRTYVPADDYRVLGTDGFGRSDSREMLRHHFEVDA SYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA* aceF MAIEIKVPDIGADEVEITEILVKVGDKVEAEQSLITVEGDKASMEVP SEQ ID SPQAGIVKEIKVSVGDKTQTGALIMIFDSADGAADAAPAQAEEKKEA NO: 39 APAAAPAAAAAKDVNVPDIGSDEVEVTEILVKVGDKVEAEQSLITVE GDKASMEVPAPFAGTVKEIKVNVGDKVSTGSLIMVFEVAGEAGAAAP AAKQEAAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKVGDKVAAE QSLITVEGDKASMEVPAPFAGVVKELKVNVGDKVKTGSLIMIFEVEG AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKAEGKSEFAENDAYVHA TPLIRRLAREFGVNLAKVKGTGRKGRILREDVQAYVKEAIKRAEAAP AATGGGIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLSRNWVMI PHVTHFDKTDITELEAFRKQQNEEAAKRKLDVKITPVVFIMKAVAAA LEQMPRFNSSLSEDGQRLTLKKYINIGVAVDIPNGLVVPVFKDVNKK GIIELSRELMTISKKARDGKLTAGEMQGGCFTISSIGGLGTTHFAPI VNAPEVAILGVSKSAMEPVWNGKEFVPRLMLPISLSFDHRVIDGADG ARFITIINNTLSDIRRLVM* Lpd MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLN SEQ ID VGCIPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVI NO: 40 NQLTGGLAGMAKGRKVKVVMGLGKFTGANTLEVEGENGKTVINFDNA IIAAGSRPIQLPFIPHEDPRIWDSTDALELKEVPERLLVMGGGIIGL EMGTVYHALGSQIDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLE TKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGKNLDA GKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGH VAAEVIAGKKHYFDPKVIPSIAYTKPEVAWVGLTEKEAKEKGISYET ATFPWAASGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLG EIGLAIEMGCDAEDIALTIHAHPTLHESVGLAAEVFEGSITDLPNPK AKKK* tesB MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFGGQVVGQALYAA SEQ ID KETVPEERLVHSFHSYFLRPGDSKKPIIYDVETLRDGNSFSARRVAA NO: 41 IQNGKPIFYMTASFQAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLA HLLPPVLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQVWIRANGSV PDDLRVHQYLLGYASDLNFLPVALQPHGIGFLEPGIQIATIDHSMWF HRPFNLNEWLLYSVESTSASSARGFVRGEFYTQDGVLVASTVQEGVM RNKN* acuI MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYKDALAI SEQ ID TGKAPVVRRFPMVPGIDFTGTVAQSSHADFKPGDRVILNGWGVGEKH NO: 42 WGGLAERARVRGDWLVPLPAPLDLRQAAMIGTAGYTAMLCVLALERH GVVPGNGEIVVSGAAGGVGSVATTLLAAKGYEVAAVTGRASEAEYLR GLGAASVIDRNELTGKVRPLGQERWAGGIDVAGSTVLANMLSMMKYR GVVAACGLAAGMDLPASVAPFILRGMTLAGVDSVMCPKTDRLAAWAR LASDLDPAKLEEMTTELPFSEVIETAPKFLDGTVRGRIVIPVTP* Sbm MSNVQEWQQLANKELSRREKTVDSLVHQTAEGIAIKPLYTEADLDNL SEQ ID EVTGTLPGLPPYVRGPRATMYTAQPWTIRQYAGFSTAKESNAFYRRN NO: 43 LAAGQKGLSVAFDLATHRGYDSDNPRVAGDVGKAGVAIDTVEDMKVL FDQIPLDKMSVSMTMNGAVLPVLAFYIVAAEEQGVTPDKLTGTIQND ILKEYLCRNTYIYPPKPSMRIIADIIAWCSGNMPRFNTISISGYHMG EAGANCVQQVAFTLADGIEYIKAAISAGLKIDDFAPRLSFFFGIGMD LFMNVAMLRAARYLWSEAVSGFGAQDFKSLALRTHCQTSGWSLTEQD PYNNVIRTTIEALAATLGGTQSLHTNAFDEALGLPTDFSARIARNTQ IIIQEESELCRTVDPLAGSYYIESLTDQIVKQARAIIQQIDEAGGMA KAIEAGLPKRMIEEASAREQSLIDQGKRVIVGVNKYKLDHEDETDVL EIDNVMVRNEQIASLERIRATRDDAAVTAALMALTHAAQHNENLLAA AVNAARVRATLGEISDALEVAFDRYLVPSQCVTGVIAQSYHQSEKSA SEFDATVAQTEQFLADNGRRPRILIAKMGQDGHDRGAKVIASAYSDL GFDVDLSPMFSTPEEIARLAVENDVHVVGASSLAAGHKTLIPELVEA LKKWGREDICVVAGGVIPPQDYAFLQERGVAAIYGPGTPMLDSVRDV LNLISQHHD* ygfD MINEATLAESIRRLRQGERATLAQAMTLVESRHPRHQALSTQLLDAI SEQ ID MPYCGNTLRLGVTGTPGAGKSTFLEAFGMLLIREGLKVAVIAVDPSS NO: 44 PVTGGSILGDKTRMNDLARAEAAFIRPVPSSGHLGGASQRARELMLL CEAAGYDVVIVETVGVGQSETEVARMVDCFISLQIAGGGDDLQGIKK GLMEVADLIVINKDDGDNHTNVAIARHMYESALHILRRKYDEWQPRV LTCSALEKRGIDEIWHAIIDFKTALTASGRLQQVRQQQSVEWLRKQT EEEVLNHLFANEDFDRYYRQTLLAVKNNTLSPRTGLRQLSEFIQTQY FD* ygfG MSYQYVNVVTINKVAVIEFNYGRKLNALSKVFIDDLMQALSDLNRPE SEQ ID IRCIILRAPSGSKVFSAGHDIHELPSGGRDPLSYDDPLRQITRMIQK NO: 45 FPKPIISMVEGSVWGGAFEMIMSSDLIIAASTSTFSMTPVNLGVPYN LVGIHNLTRDAGFHIVKELIFTASPITAQRALAVGILNHVVEVEELE DFTLQMAHHISEKAPLAIAVIKEELRVLGEAHTMNSDEFERIQGMRR AVYDSEDYQEGMNAFLEKRKPNFVGH* yghH METQWTRMTANEAAEIIQHNDMVAFSGFTPAGSPKALPTAIARRANE SEQ ID QHEAKKPYQIRLLTGASISAAADDVLSDADAVSNRAPYQTSSGLRKK NO: 46 INQGAVSFVDLHLSEVAQMVNYGFFGDIDVAVIEASALAPDGRVWLT SGIGMAPTVVLLRAKKVHELNHYHDPRVAELADIVIPGAPPRRNSVS IFHAMDRVGTRYVQIDPKKIVAVVETNLPDAGNMLDKQNPMCQQIAD NVVTFLLQEMAHGRIPPEFLPLQSGVGNINNAVMARLGENPVIPPFM MYSEVLQESVVHLLETGKISGASASSLTISADSLRKIYDNMDYFASR IVLRPQEISNNPEIIRRLGVIALNVGLEFDIYGHANSTHVAGVDLMN GIGGSGDFERNAYLSIFMAPSIAKEGKISTVVPMCSRVDHSEHSVKV IITEQGIADLRGLSPLQRARTIIDNCAHPMYRDYLHRYLENAPGGHI HHDLSHVFDLHRNLIATGSMLG* mutA MSSTDQGTNPADTDDLTPTTLSLAGDFPKATEEQWEREVEKVFNRGRP SEQ ID PEKQLTFAECLKRLTVHTVDGIDIVPMYRPKDAPKKLGYPGVTPFTRG NO: 47 TTVRNGDMDAWDVRALHEDPDEKFTRKAILEDLERGVTSLLLRVDPDA IAPEHLDEVLSDVLLEMTKVEVFSRYDQGAAAEALMGVYERSDKPAKD LALNLGLDPIGFAALQGTEPDLTVLGDWVRRLAKFSPDSRAVTIDANV YHNAGAGDVAELAWALATGAEYVRALVEQGFNATEAFDTINFRVTATH DQFLTIARLRALREAWARIGEVFGVDEDKRGARQNAITSWRELTREDP YVNILRGSIATFSASVGGAESITTLPFTQALGLPEDDFPLRIARNTGI VLAEEVNIGRVNDPAGGSYYVESLTRTLADAAWKEFQEVEKLGGMSKA VMTEHVTKVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFP TAPARKGLAWHRDSEVFEQLMDRSTSVSERPKVFLACLGTRRDFGGRE GFSSPVWHIAGIDTPQVEGGTTAEIVEAFKKSGAQVADLCSSAKIYAQ QGLEVAKALKAAGAKALYLSGAFKEFGDDAAEAEKLIDGRLYMGMDVV DTLSSTLDILGVAK mutB SEQ ID VSTLPRFDSVDLGNAPVPADAAQRFEELAAKAGTEEAWETAEQIPVGT NO: 48 LFNEDVYKDMDWLDTYAGIPPFVHGPYATMYAFRPWTIRQYAGFSTAK ESNAFYRRNLAAGQKGLSVAFDLPTHRGYDSDNPRVAGDVGMAGVAID SIYDMRELFAGIPLDQMSVSMTMNGAVLPILALYVVTAEEQGVKPEQL AGTIQNDILKEFMVRNTYIYPPQPSMRIISEIFAYTSANMPKWNSISI SGYHMQEAGATADIEMAYTLADGVDYIRAGESVGLNVDQFAPRLSFFW GIGMNFFMEVAKLRAARMLWAKLVHQFGPKNPKSMSLRTHSQTSGWSL TAQDVYNNVVRTCIEAMAATQGHTQSLHTNSLDEAIALPTDFSARIAR NTQLFLQQESGTTRVIDPWSGSAYVEELTWDLARKAWGHIQEVEKVGG MAKAIEKGIPKMRIEEAAARTQARIDSGRQPLIGVNKYRLEHEPPLDV LKVDNSTVLAEQKAKLVKLRAERDPEKVKAALDKITWAAANPDDKDPD RNLLKLCIDAGRAMATVGEMSDALEKVFGRYTAQIRTISGVYSKEVKN TPEVEEARELVEEFEQAEGRRPRILLAKMGQDGHDRGQKVIATAYADL GFDVDVGPLFQTPEETARQAVEADVHVVGVSSLAGGHLTLVPALRKEL DKLGRPDILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLVK KLRASLDA GI:18042134 MSNEDLFICIDHVAYACPDADEASKYYQETFGWHELHREENPEQGVVE SEQ ID IMMAPAAKLTEHMTQVQVMAPLNDESTVAKWLAKHNGRAGLHHMAWRV NO: 49 DDIDAVSATLRERGVQLLYDEPKLGTGGNRINFMHPKSGKGVLIELTQ YPKN mmdA MAENNNLKLASTMEGRVEQLAEQRQVIEAGGGERRVEKQHSQGKQTAR SEQ ID ERLNNLLDPHSFDEVGAFRKHRTTLFGMDKAVVPADGVVTGRGTILGR NO: 50 PVHAASQDFTVMGGSAGETQSTKVVETMEQALLTGTPFLFFYDSGGAR IQEGIDSLSGYGKMFFANVKLSGVVPQIAIIAGPCAGGASYSPALTDF IIMTKKAHMFITGPQVIKSVTGEDVTADELGGAEAHMAISGNIHFVAE DDDAAELIAKKLLSFLPQNNTEEASFVNPNNDVSPNTELRDIVPIDGK KGYDVRDVIAKIVDWGDYLEVKAGYATNLVTAFARVNGRSVGIVANQP SVMSGCLDINASDKAAEFVNFCDSFNIPLVQLVDVPGFLPGVQQEYGG IIRHGAKMLYAYSEATVPKITVVLRKAYGGSYLAMCNRDLGADAVYAW PSAEIAVMGAEGAANVIFRKEIKAADDPDAMRAEKIEEYQNAFNTPYV AAARGQVDDVIDPADTRRKIASALEMYATKRQTRPAKKHGNFPC PFREUD_ MSPREIEVSEPREVGITELVLRDAHQSLMATRMAMEDMVGACADIDAA 18870 GYWSVECWGGATYDSCIRFLNEDPWERLRTFRKLMPNSRLQMLLRGQN SEQ ID LLGYRHYNDEVVDRFVDKSAENGMDVFRVFDAMNDPRNMAHAMAAVKK NO: 51 AGKHAQGTICYTISPVHTVEGYVKLAGQLLDMGADSIALKDMAALLKP QPAYDIIKAIKDTYGQKTQINLHCHSTTGVTEVSLMKAIEAGVDVVDT AISSMSLGPGHNPTESVAEMLEGTGYTTNLDYDRLHKIRDHFKAIRPK YKKFESKTLVDTSIFKSQIPGGMLSNMESQLRAQGAEDKMDEVMAEVP RVRKAAGFPPLVTPSSQIVGTQAVFNVMMGEYKRMTGEFADIMLGYYG ASPADRDPKVVKLAEEQSGKKPITQRPADLLPPEWEEQSKEAAALKGF NGTDEDVLTYALFPQVAPVFFEHRAEGPHSVALTDAQLKAEAEGDEKS LAVAGPVTYNVNVGGTVREVTVQQA Bccp MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPRAAGGAG SEQ ID AGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINA NO: 52 PTDGKVEKVLVKERDAVQGGQGLIKIG

In some embodiments, the genetically engineered bacteria encode one or more polypeptide sequences of Table 5 (SEQ ID NO: 27-SEQ ID NO: 52) or a functional fragment or variant thereof. In some embodiments, genetically engineered bacteria comprise a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the polypeptide sequence of one or more polypeptide sequence of Table 5 (SEQ ID NO: 27-SEQ ID NO: 52) or a functional fragment thereof.

In one embodiment, the bacterial cell comprises a heterologous propionate gene cassette. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous propionate gene cassette operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a propionate gene cassette from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a propionate gene cassette. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a propionate gene cassette, as well as at least one copy of a propionate gene cassette from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a propionate gene cassette. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a propionate gene cassette.

Multiple distinct propionate gene cassettes are known in the art. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a non-bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, the gene encoding the propionate gene cassette is derived from an organism of the genus or species that includes, but is not limited to, Clostridium propionicum, Megasphaera elsdenii, or Prevotella ruminicola.

In one embodiment, the propionate gene cassette has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the propionate gene cassette has been codon-optimized for use in Escherichia coli. In another embodiment, the propionate gene cassette has been codon-optimized for use in Lactococcus. When the propionate gene cassette is expressed in the engineered bacterial cells, the bacterial cells produce more propionate than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous propionate gene cassette may be used to generate propionate to treat liver disease, such as nonalcoholic steatohepatitis (NASH).

The present disclosure further comprises genes encoding functional fragments of propionate biosynthesis enzymes or functional variants of a propionate biosynthesis enzyme. As used herein, the term “functional fragment thereof” or “functional variant thereof” relates to an element having qualitative biological activity in common with the wild-type enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated propionate biosynthesis enzyme is one which retains essentially the same ability to synthesize propionate as the propionate biosynthesis enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having propionate biosynthesis enzyme activity may be truncated at the N-terminus or C-terminus, and the retention of propionate biosynthesis enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional variant. In another embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional fragment.

As used herein, the term “percent (%) sequence identity” or “percent (%) identity,” also including “homology,” is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The present disclosure encompasses propionate biosynthesis enzymes comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).

In some embodiments, a propionate biosynthesis enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the propionate biosynthesis enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

In one embodiment, the propionate biosynthesis gene cassette is from Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium propionicum. In another embodiment, the propionate biosynthesis gene cassette is from a Megasphaera spp. In one embodiment, the Megasphaera spp. is Megasphaera elsdenii. In another embodiment, the propionate biosynthesis gene cassette is from Prevotella spp. In one embodiment, the Prevotella spp. is Prevotella ruminicola. Other propionate biosynthesis gene cassettes are well-known to one of ordinary skill in the art.

In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA^(fbr), thrB, thrC, ilvA^(fbr), aceE, aceF, and lpd, and optionally further comprise tesB. The genes may be codon-optimized, and translational and transcriptional elements may be added.

In one embodiment, the pct gene has at least about 80% identity with SEQ ID NO: 1. In another embodiment, the pct gene has at least about 85% identity with SEQ ID NO: 1. In one embodiment, the pct gene has at least about 90% identity with SEQ ID NO: 1. In one embodiment, the pct gene has at least about 95% identity with SEQ ID NO: 1. In another embodiment, the pct gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. Accordingly, in one embodiment, the pct gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In another embodiment, the pct gene comprises the sequence of SEQ ID NO: 1. In yet another embodiment the pct gene consists of the sequence of SEQ ID NO: 1.

In one embodiment, the lcdA gene has at least about 80% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene has at least about 85% identity with SEQ ID NO: 2. In one embodiment, the lcdA gene has at least about 90% identity with SEQ ID NO: 2. In one embodiment, the lcdA gene has at least about 95% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene comprises the sequence of SEQ ID NO: 2. In yet another embodiment the lcdA gene consists of the sequence of SEQ ID NO: 2.

In one embodiment, the lcdB gene has at least about 80% identity with SEQ ID NO: 3. In another embodiment, the lcdB gene has at least about 85% identity with SEQ ID NO: 3. In one embodiment, the lcdB gene has at least about 90% identity with SEQ ID NO: 3. In one embodiment, the lcdB gene has at least about 95% identity with SEQ ID NO: 3. In another embodiment, the lcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. Accordingly, in one embodiment, the lcdB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In another embodiment, the lcdB gene comprises the sequence of SEQ ID NO: 3. In yet another embodiment the lcdB gene consists of the sequence of SEQ ID NO: 3.

In one embodiment, the lcdC gene has at least about 80% identity with SEQ ID NO: 4. In another embodiment, the lcdC gene has at least about 85% identity with SEQ ID NO: 4. In one embodiment, the lcdC gene has at least about 90% identity with SEQ ID NO: 4. In one embodiment, the lcdC gene has at least about 95% identity with SEQ ID NO: 4. In another embodiment, the lcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In another embodiment, the lcdC gene comprises the sequence of SEQ ID NO: 4. In yet another embodiment the lcdC gene consists of the sequence of SEQ ID NO: 4.

In one embodiment, the e0 gene has at least about 80% identity with SEQ ID NO: 5. In another embodiment, the e0 gene has at least about 85% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 90% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 95% identity with SEQ ID NO: 5. In another embodiment, the etfA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. Accordingly, in one embodiment, the etfA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In another embodiment, the e0 gene comprises the sequence of SEQ ID NO: 5. In yet another embodiment the etfA gene consists of the sequence of SEQ ID NO: 5.

In one embodiment, the acrB gene has at least about 80% identity with SEQ ID NO: 6. In another embodiment, the acrB gene has at least about 85% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 90% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 95% identity with SEQ ID NO: 6. In another embodiment, the acrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. Accordingly, in one embodiment, the acrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO: 6. In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO: 6.

In one embodiment, the acrC gene has at least about 80% identity with SEQ ID NO: 7. In another embodiment, the acrC gene has at least about 85% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 90% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 95% identity with SEQ ID NO: 7. In another embodiment, the acrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. Accordingly, in one embodiment, the acrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO: 7. In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO: 7.

In one embodiment, the thrA^(fbr) gene has at least about 80% identity with SEQ ID NO: 8. In another embodiment, the thrA^(fbr) gene has at least about 85% identity with SEQ ID NO: 8. In one embodiment, the thrA^(fbr) gene has at least about 90% identity with SEQ ID NO: 8. In one embodiment, the thrA^(fbr) gene has at least about 95% identity with SEQ ID NO: 8. In another embodiment, the thrA^(fbr) gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. Accordingly, in one embodiment, the thrg^(br) gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. In another embodiment, the thrA^(fbr) gene comprises the sequence of SEQ ID NO: 8. In yet another embodiment the thrA^(fbr) gene consists of the sequence of SEQ ID NO: 8.

In one embodiment, the thrB gene has at least about 80% identity with SEQ ID NO: 9. In another embodiment, the thrB gene has at least about 85% identity with SEQ ID NO: 9. In one embodiment, the thrB gene has at least about 90% identity with SEQ ID NO: 9. In one embodiment, the thrB gene has at least about 95% identity with SEQ ID NO: 9. In another embodiment, the thrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. Accordingly, in one embodiment, the thrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. In another embodiment, the thrB gene comprises the sequence of SEQ ID NO: 9. In yet another embodiment the thrB gene consists of the sequence of SEQ ID NO: 9.

In one embodiment, the thrC gene has at least about 80% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 85% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 90% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 95% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the thrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the thrC gene consists of the sequence of SEQ ID NO: 10.

In one embodiment, the ilvA^(fbr) gene has at least about 80% identity with SEQ ID NO: 11. In another embodiment, the ilvA^(fbr) gene has at least about 85% identity with SEQ ID NO: 11. In one embodiment, the ilvA^(fbr) gene has at least about 90% identity with SEQ ID NO: 11. In one embodiment, the ilvA^(fbr) gene has at least about 95% identity with SEQ ID NO: 11. In another embodiment, the ilvA^(fbr) gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. Accordingly, in one embodiment, the ilvA^(fbr) gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. In another embodiment, the ilvA^(fbr) gene comprises the sequence of SEQ ID NO: 11. In yet another embodiment the ilvA^(fbr) gene consists of the sequence of SEQ ID NO: 11.

In one embodiment, the aceE gene has at least about 80% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 85% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 90% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 95% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. Accordingly, in one embodiment, the aceE gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO: 12. In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO: 12.

In one embodiment, the aceF gene has at least about 80% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 85% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 90% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 95% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. Accordingly, in one embodiment, the aceF gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO: 13. In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO: 13.

In one embodiment, the lpd gene has at least about 80% identity with SEQ ID NO: 14. In another embodiment, the lpd gene has at least about 85% identity with SEQ ID NO: 14. In one embodiment, the lpd gene has at least about 90% identity with SEQ ID NO: 14. In one embodiment, the lpd gene has at least about 95% identity with SEQ ID NO: 14. In another embodiment, the lpd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14. Accordingly, in one embodiment, the lpd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14. In another embodiment, the lpd gene comprises the sequence of SEQ ID NO: 14. In yet another embodiment the lpd gene consists of the sequence of SEQ ID NO: 14.

In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 15.

In one embodiment, the acuI gene has at least about 80% identity with SEQ ID NO: 16. In another embodiment, the acuI gene has at least about 85% identity with SEQ ID NO: 16. In one embodiment, the acuI gene has at least about 90% identity with SEQ ID NO: 16. In one embodiment, the acuI gene has at least about 95% identity with SEQ ID NO: 16. In another embodiment, the acuI gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16. Accordingly, in one embodiment, the acuI gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16. In another embodiment, the acuI gene comprises the sequence of SEQ ID NO: 16. In yet another embodiment the acuI gene consists of the sequence of SEQ ID NO: 16.

In one embodiment, the sbm gene has at least about 80% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.0. Accordingly, in one embodiment, the sbm gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17. In another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 17. In yet another embodiment the sbm gene consists of the sequence of SEQ ID NO: 17.

In one embodiment, the ygfD gene has at least about 80% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 85% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 90% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 95% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18.. Accordingly, in one embodiment, the ygfD gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO: 18. In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO: 18.

In one embodiment, the ygfG gene has at least about 80% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 85% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 90% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 95% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19.. Accordingly, in one embodiment, the ygfG gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO: 19. In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO: 19.

In one embodiment, the ygfH gene has at least about 80% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 85% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 90% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 95% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20.. Accordingly, in one embodiment, the ygfH gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO: 20. In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO: 20.

In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of or or more of SEQ ID NO: 27 through SEQ ID NO: 52.

In some embodiments, one or more of the propionate biosynthesis genes is a synthetic propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is an E. coli propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C. glutamicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C. propionicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a R. sphaeroides propionate biosynthesis gene. The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate.

In some embodiments, the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing propionate. In some embodiments, one or more of the propionate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase propionate production. In some embodiments, the local production of propionate reduces food intake and ameliorates metabolic disease (Lin et al., 2012). In some embodiments, the genetically engineered bacteria are capable of expressing the propionate biosynthesis cassette and producing propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In one embodiment, the propionate gene cassette is directly operably linked to a first promoter. In another embodiment, the propionate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the propionate gene cassette is operably linked to a promoter that it is not naturally linked to in nature.

In some embodiments, the propionate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the propionate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the propionate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.

The propionate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the propionate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the propionate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located on a plasmid in the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.

In some embodiments, the propionate gene cassette is expressed on a low-copy plasmid. In some embodiments, the propionate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of propionate.

Butyrate

In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of butyrogenic genes (see, e.g., Table 3). Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. In some embodiments, the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the eight genes of the butyrate biosynthesis pathway from Peptoclostridium difficile, e.g., Peptoclostridium difficile strain 630: bcd2, eff133, etfA3, thiAl, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013) and are capable of producing butyrate. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. In some embodiments, the genetically engineered bacteria comprise a combination of butyrogenic genes from different species, strains, and/or substrains of bacteria and are capable of producing butyrate. For example, in some embodiments, the genetically engineered bacteria comprise bcd2, etfl33, etfA3, and thiA1 from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB. In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

In some embodiments, additional genes may be mutated or knocked out, to further increase the levels of butyrate production. Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization. Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.

Table 6 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes.

TABLE 6 Exemplary Butyrate Cassette Sequences Description Sequence bcd2 ATGGATTTAAATTCTAAAAAATATCAGATGCTTAAAGAGCTATA SEQ ID NO: 53 TGTAAGCTTCGCTGAAAATGAAGTTAAACCTTTAGCAACAGAAC TTGATGAAGAAGAAAGATTTCCTTATGAAACAGTGGAAAAAATG GCAAAAGCAGGAATGATGGGTATACCATATCCAAAAGAATATGG TGGAGAAGGTGGAGACACTGTAGGATATATAATGGCAGTTGAAG AATTGTCTAGAGTTTGTGGTACTACAGGAGTTATATTATCAGCT CATACATCTCTTGGCTCATGGCCTATATATCAATATGGTAATGA AGAACAAAAACAAAAATTCTTAAGACCACTAGCAAGTGGAGAAA AATTAGGAGCATTTGGTCTTACTGAGCCTAATGCTGGTACAGAT GCGTCTGGCCAACAAACAACTGCTGTTTTAGACGGGGATGAATA CATACTTAATGGCTCAAAAATATTTATAACAAACGCAATAGCTG GTGACATATATGTAGTAATGGCAATGACTGATAAATCTAAGGGG AACAAAGGAATATCAGCATTTATAGTTGAAAAAGGAACTCCTGG GTTTAGCTTTGGAGTTAAAGAAAAGAAAATGGGTATAAGAGGTT CAGCTACGAGTGAATTAATATTTGAGGATTGCAGAATACCTAAA GAAAATTTACTTGGAAAAGAAGGTCAAGGATTTAAGATAGCAAT GTCTACTCTTGATGGTGGTAGAATTGGTATAGCTGCACAAGCTT TAGGTTTAGCACAAGGTGCTCTTGATGAAACTGTTAAATATGTA AAAGAAAGAGTACAATTTGGTAGACCATTATCAAAATTCCAAAA TACACAATTCCAATTAGCTGATATGGAAGTTAAGGTACAAGCGG CTAGACACCTTGTATATCAAGCAGCTATAAATAAAGACTTAGGA AAACCTTATGGAGTAGAAGCAGCAATGGCAAAATTATTTGCAGC TGAAACAGCTATGGAAGTTACTACAAAAGCTGTACAACTTCATG GAGGATATGGATACACTCGTGACTATCCAGTAGAAAGAATGATG AGAGATGCTAAGATAACTGAAATATATGAAGGAACTAGTGAAGT TCAAAGAATGGTTATTTCAGGAAAACTATTAAAATAG etfB3 ATGAATATAGTCGTTTGTATAAAACAAGTTCCAGATACAACAGA SEQ ID NO: 54 AGTTAAACTAGATCCTAATACAGGTACTTTAATTAGAGATGGAG TACCAAGTATAATAAACCCTGATGATAAAGCAGGTTTAGAAGAA GCTATAAAATTAAAAGAAGAAATGGGTGCTCATGTAACTGTTAT AACAATGGGACCTCCTCAAGCAGATATGGCTTTAAAAGAAGCTT TAGCAATGGGTGCAGATAGAGGTATATTATTAACAGATAGAGCA TTTGCGGGTGCTGATACTTGGGCAACTTCATCAGCATTAGCAGG AGCATTAAAAAATATAGATTTTGATATTATAATAGCTGGAAGAC AGGCGATAGATGGAGATACTGCACAAGTTGGACCTCAAATAGCT GAACATTTAAATCTTCCATCAATAACATATGCTGAAGAAATAAA AACTGAAGGTGAATATGTATTAGTAAAAAGACAATTTGAAGATT GTTGCCATGACTTAAAAGTTAAAATGCCATGCCTTATAACAACT CTTAAAGATATGAACACACCAAGATACATGAAAGTTGGAAGAAT ATATGATGCTTTCGAAAATGATGTAGTAGAAACATGGACTGTAA AAGATATAGAAGTTGACCCTTCTAATTTAGGTCTTAAAGGTTCT CCAACTAGTGTATTTAAATCATTTACAAAATCAGTTAAACCAGC TGGTACAATATACAATGAAGATGCGAAAACATCAGCTGGAATTA TCATAGATAAATTAAAAGAGAAGTATATCATATAA etfA3 ATGGGTAACGTTTTAGTAGTAATAGAACAAAGAGAAAATGTAAT SEQ ID NO: 55 TCAAACTGTTTCTTTAGAATTACTAGGAAAGGCTACAGAAATAG CAAAAGATTATGATACAAAAGTTTCTGCATTACTTTTAGGTAGT AAGGTAGAAGGTTTAATAGATACATTAGCACACTATGGTGCAGA TGAGGTAATAGTAGTAGATGATGAAGCTTTAGCAGTGTATACAA CTGAACCATATACAAAAGCAGCTTATGAAGCAATAAAAGCAGCT GACCCTATAGTTGTATTATTTGGTGCAACTTCAATAGGTAGAGA TTTAGCGCCTAGAGTTTCTGCTAGAATACATACAGGTCTTACTG CTGACTGTACAGGTCTTGCAGTAGCTGAAGATACAAAATTATTA TTAATGACAAGACCTGCCTTTGGTGGAAATATAATGGCAACAAT AGTTTGTAAAGATTTCAGACCTCAAATGTCTACAGTTAGACCAG GGGTTATGAAGAAAAATGAACCTGATGAAACTAAAGAAGCTGTA ATTAACCGTTTCAAGGTAGAATTTAATGATGCTGATAAATTAGT TCAAGTTGTACAAGTAATAAAAGAAGCTAAAAAACAAGTTAAAA TAGAAGATGCTAAGATATTAGTTTCTGCTGGACGTGGAATGGGT GGAAAAGAAAACTTAGACATACTTTATGAATTAGCTGAAATTAT AGGTGGAGAAGTTTCTGGTTCTCGTGCCACTATAGATGCAGGTT GGTTAGATAAAGCAAGACAAGTTGGTCAAACTGGTAAAACTGTA AGACCAGACCTTTATATAGCATGTGGTATATCTGGAGCAATACA ACATATAGCTGGTATGGAAGATGCTGAGTTTATAGTTGCTATAA ATAAAAATCCAGAAGCTCCAATATTTAAATATGCTGATGTTGGT ATAGTTGGAGATGTTCATAAAGTGCTTCCAGAACTTATCAGTCA GTTAAGTGTTGCAAAAGAAAAAGGTGAAGTTTTAGCTAACTAA thiA1 ATGAGAGAAGTAGTAATTGCCAGTGCAGCTAGAACAGCAGTAGG SEQ ID NO: 56 AAGTTTTGGAGGAGCATTTAAATCAGTTTCAGCGGTAGAGTTAG GGGTAACAGCAGCTAAAGAAGCTATAAAAAGAGCTAACATAACT CCAGATATGATAGATGAATCTCTTTTAGGGGGAGTACTTACAGC AGGTCTTGGACAAAATATAGCAAGACAAATAGCATTAGGAGCAG GAATACCAGTAGAAAAACCAGCTATGACTATAAATATAGTTTGT GGTTCTGGATTAAGATCTGTTTCAATGGCATCTCAACTTATAGC ATTAGGTGATGCTGATATAATGTTAGTTGGTGGAGCTGAAAACA TGAGTATGTCTCCTTATTTAGTACCAAGTGCGAGATATGGTGCA AGAATGGGTGATGCTGCTTTTGTTGATTCAATGATAAAAGATGG ATTATCAGACATATTTAATAACTATCACATGGGTATTACTGCTG AAAACATAGCAGAGCAATGGAATATAACTAGAGAAGAACAAGAT GAATTAGCTCTTGCAAGTCAAAATAAAGCTGAAAAAGCTCAAGC TGAAGGAAAATTTGATGAAGAAATAGTTCCTGTTGTTATAAAAG GAAGAAAAGGTGACACTGTAGTAGATAAAGATGAATATATTAAG CCTGGCACTACAATGGAGAAACTTGCTAAGTTAAGACCTGCATT TAAAAAAGATGGAACAGTTACTGCTGGTAATGCATCAGGAATAA ATGATGGTGCTGCTATGTTAGTAGTAATGGCTAAAGAAAAAGCT GAAGAACTAGGAATAGAGCCTCTTGCAACTATAGTTTCTTATGG AACAGCTGGTGTTGACCCTAAAATAATGGGATATGGACCAGTTC CAGCAACTAAAAAAGCTTTAGAAGCTGCTAATATGACTATTGAA GATATAGATTTAGTTGAAGCTAATGAGGCATTTGCTGCCCAATC TGTAGCTGTAATAAGAGACTTAAATATAGATATGAATAAAGTTA ATGTTAATGGTGGAGCAATAGCTATAGGACATCCAATAGGATGC TCAGGAGCAAGAATACTTACTACACTTTTATATGAAATGAAGAG AAGAGATGCTAAAACTGGTCTTGCTACACTTTGTATAGGCGGTG GAATGGGAACTACTTTAATAGTTAAGAGATAG hbd ATGAAATTAGCTGTAATAGGTAGTGGAACTATGGGAAGTGGTAT SEQ ID NO: 57 TGTACAAACTTTTGCAAGTTGTGGACATGATGTATGTTTAAAGA GTAGAACTCAAGGTGCTATAGATAAATGTTTAGCTTTATTAGAT AAAAATTTAACTAAGTTAGTTACTAAGGGAAAAATGGATGAAGC TACAAAAGCAGAAATATTAAGTCATGTTAGTTCAACTACTAATT ATGAAGATTTAAAAGATATGGATTTAATAATAGAAGCATCTGTA GAAGACATGAATATAAAGAAAGATGTTTTCAAGTTACTAGATGA ATTATGTAAAGAAGATACTATCTTGGCAACAAATACTTCATCAT TATCTATAACAGAAATAGCTTCTTCTACTAAGCGCCCAGATAAA GTTATAGGAATGCATTTCTTTAATCCAGTTCCTATGATGAAATT AGTTGAAGTTATAAGTGGTCAGTTAACATCAAAAGTTACTTTTG ATACAGTATTTGAATTATCTAAGAGTATCAATAAAGTACCAGTA GATGTATCTGAATCTCCTGGATTTGTAGTAAATAGAATACTTAT ACCTATGATAAATGAAGCTGTTGGTATATATGCAGATGGTGTTG CAAGTAAAGAAGAAATAGATGAAGCTATGAAATTAGGAGCAAAC CATCCAATGGGACCACTAGCATTAGGTGATTTAATCGGATTAGA TGTTGTTTTAGCTATAATGAACGTTTTATATACTGAATTTGGAG ATACTAAATATAGACCTCATCCACTTTTAGCTAAAATGGTTAGA GCTAATCAATTAGGAAGAAAAACTAAGATAGGATTCTATGATTA TAATAAATAA crt2 ATGAGTACAAGTGATGTTAAAGTTTATGAGAATGTAGCTGTTGA SEQ ID NO: 58 AGTAGATGGAAATATATGTACAGTGAAAATGAATAGACCTAAAG CCCTTAATGCAATAAATTCAAAGACTTTAGAAGAACTTTATGAA GTATTTGTAGATATTAATAATGATGAAACTATTGATGTTGTAAT ATTGACAGGGGAAGGAAAGGCATTTGTAGCTGGAGCAGATATTG CATACATGAAAGATTTAGATGCTGTAGCTGCTAAAGATTTTAGT ATCTTAGGAGCAAAAGCTTTTGGAGAAATAGAAAATAGTAAAAA AGTAGTGATAGCTGCTGTAAACGGATTTGCTTTAGGTGGAGGAT GTGAACTTGCAATGGCATGTGATATAAGAATTGCATCTGCTAAA GCTAAATTTGGTCAGCCAGAAGTAACTCTTGGAATAACTCCAGG ATATGGAGGAACTCAAAGGCTTACAAGATTGGTTGGAATGGCAA AAGCAAAAGAATTAATCTTTACAGGTCAAGTTATAAAAGCTGAT GAAGCTGAAAAAATAGGGCTAGTAAATAGAGTCGTTGAGCCAGA CATTTTAATAGAAGAAGTTGAGAAATTAGCTAAGATAATAGCTA AAAATGCTCAGCTTGCAGTTAGATACTCTAAAGAAGCAATACAA CTTGGTGCTCAAACTGATATAAATACTGGAATAGATATAGAATC TAATTTATTTGGTCTTTGTTTTTCAACTAAAGACCAAAAAGAAG GAATGTCAGCTTTCGTTGAAAAGAGAGAAGCTAACTTTATAAAA GGGTAA pbt ATGAGAAGTTTTGAAGAAGTAATTAAGTTTGCAAAAGAAAGAGG SEQ ID NO: 59 ACCTAAAACTATATCAGTAGCATGTTGCCAAGATAAAGAAGTTT TAATGGCAGTTGAAATGGCTAGAAAAGAAAAAATAGCAAATGCC ATTTTAGTAGGAGATATAGAAAAGACTAAAGAAATTGCAAAAAG CATAGACATGGATATCGAAAATTATGAACTGATAGATATAAAAG ATTTAGCAGAAGCATCTCTAAAATCTGTTGAATTAGTTTCACAA GGAAAAGCCGACATGGTAATGAAAGGCTTAGTAGACACATCAAT AATACTAAAAGCAGTTTTAAATAAAGAAGTAGGTCTTAGAACTG GAAATGTATTAAGTCACGTAGCAGTATTTGATGTAGAGGGATAT GATAGATTATTTTTCGTAACTGACGCAGCTATGAACTTAGCTCC TGATACAAATACTAAAAAGCAAATCATAGAAAATGCTTGCACAG TAGCACATTCATTAGATATAAGTGAACCAAAAGTTGCTGCAATA TGCGCAAAAGAAAAAGTAAATCCAAAAATGAAAGATACAGTTGA AGCTAAAGAACTAGAAGAAATGTATGAAAGAGGAGAAATCAAAG GTTGTATGGTTGGTGGGCCTTTTGCAATTGATAATGCAGTATCT TTAGAAGCAGCTAAACATAAAGGTATAAATCATCCTGTAGCAGG ACGAGCTGATATATTATTAGCCCCAGATATTGAAGGTGGTAACA TATTATATAAAGCTTTGGTATTCTTCTCAAAATCAAAAAATGCA GGAGTTATAGTTGGGGCTAAAGCACCAATAATATTAACTTCTAG AGCAGACAGTGAAGAAACTAAACTAAACTCAATAGCTTTAGGTG TTTTAATGGCAGCAAAGGCATAA buk ATGAGCAAAATATTTAAAATCTTAACAATAAATCCTGGTTCGAC SEQ ID NO: 60 ATCAACTAAAATAGCTGTATTTGATAATGAGGATTTAGTATTTG AAAAAACTTTAAGACATTCTTCAGAAGAAATAGGAAAATATGAG AAGGTGTCTGACCAATTTGAATTTCGTAAACAAGTAATAGAAGA AGCTCTAAAAGAAGGTGGAGTAAAAACATCTGAATTAGATGCTG TAGTAGGTAGAGGAGGACTTCTTAAACCTATAAAAGGTGGTACT TATTCAGTAAGTGCTGCTATGATTGAAGATTTAAAAGTGGGAGT TTTAGGAGAACACGCTTCAAACCTAGGTGGAATAATAGCAAAAC AAATAGGTGAAGAAGTAAATGTTCCTTCATACATAGTAGACCCT GTTGTTGTAGATGAATTAGAAGATGTTGCTAGAATTTCTGGTAT GCCTGAAATAAGTAGAGCAAGTGTAGTACATGCTTTAAATCAAA AGGCAATAGCAAGAAGATATGCTAGAGAAATAAACAAGAAATAT GAAGATATAAATCTTATAGTTGCACACATGGGTGGAGGAGTTTC TGTTGGAGCTCATAAAAATGGTAAAATAGTAGATGTTGCAAACG CATTAGATGGAGAAGGACCTTTCTCTCCAGAAAGAAGTGGTGGA CTACCAGTAGGTGCATTAGTAAAAATGTGCTTTAGTGGAAAATA TACTCAAGATGAAATTAAAAAGAAAATAAAAGGTAATGGCGGAC TAGTTGCATACTTAAACACTAATGATGCTAGAGAAGTTGAAGAA AGAATTGAAGCTGGTGATGAAAAAGCTAAATTAGTATATGAAGC TATGGCATATCAAATCTCTAAAGAAATAGGAGCTAGTGCTGCAG TTCTTAAGGGAGATGTAAAAGCAATATTATTAACTGGTGGAATC GCATATTCAAAAATGTTTACAGAAATGATTGCAGATAGAGTTAA ATTTATAGCAGATGTAAAAGTTTATCCAGGTGAAGATGAAATGA TTGCATTAGCTCAAGGTGGACTTAGAGTTTTAACTGGTGAAGAA GAGGCTCAAGTTTATGATAACTAA ter ATGATCGTAAAACCTATGGTACGCAACAATATCTGCCTGAACGC SEQ ID NO: 61 CCATCCTCAGGGCTGCAAGAAGGGAGTGGAAGATCAGATTGAAT ATACCAAGAAACGCATTACCGCAGAAGTCAAAGCTGGCGCAAAA GCTCCAAAAAACGTTCTGGTGCTTGGCTGCTCAAATGGTTACGG CCTGGCGAGCCGCATTACTGCTGCGTTCGGATACGGGGCTGCGA CCATCGGCGTGTCCTTTGAAAAAGCGGGTTCAGAAACCAAATAT GGTACACCGGGATGGTACAATAATTTGGCATTTGATGAAGCGGC AAAACGCGAGGGTCTTTATAGCGTGACGATCGACGGCGATGCGT TTTCAGACGAGATCAAGGCCCAGGTAATTGAGGAAGCCAAAAAA AAAGGTATCAAATTTGATCTGATCGTATACAGCTTGGCCAGCCC AGTACGTACTGATCCTGATACAGGTATCATGCACAAAAGCGTTT TGAAACCCTTTGGAAAAACGTTCACAGGCAAAACAGTAGATCCG TTTACTGGCGAGCTGAAGGAAATCTCCGCGGAACCAGCAAATGA CGAGGAAGCAGCCGCCACTGTTAAAGTTATGGGGGGTGAAGATT GGGAACGTTGGATTAAGCAGCTGTCGAAGGAAGGCCTCTTAGAA GAAGGCTGTATTACCTTGGCCTATAGTTATATTGGCCCTGAAGC TACCCAAGCTTTGTACCGTAAAGGCACAATCGGCAAGGCCAAAG AACACCTGGAGGCCACAGCACACCGTCTCAACAAAGAGAACCCG TCAATCCGTGCCTTCGTGAGCGTGAATAAAGGCCTGGTAACCCG CGCAAGCGCCGTAATCCCGGTAATCCCTCTGTATCTCGCCAGCT TGTTCAAAGTAATGAAAGAGAAGGGCAATCATGAAGGTTGTATT GAACAGATCACGCGTCTGTACGCCGAGCGCCTGTACCGTAAAGA TGGTACAATTCCAGTTGATGAGGAAAATCGCATTCGCATTGATG ATTGGGAGTTAGAAGAAGACGTCCAGAAAGCGGTATCCGCGTTG ATGGAGAAAGTCACGGGTGAAAACGCAGAATCTCTCACTGACTT AGCGGGGTACCGCCATGATTTCTTAGCTAGTAACGGCTTTGATG TAGAAGGTATTAATTATGAAGCGGAAGTTGAACGCTTCGACCGT ATCTGA tesB ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA SEQ ID NO: 15 AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAG GTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTG TATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGA TTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGC GCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTA TATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATC AAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCG GAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGT GCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTC CGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCA CATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGA CCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTA ACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTC GAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGTT CCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGG AGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAG TTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGA AGGGGTGATGCGTAATCACAATTAA

Exemplary polypeptide sequences for the production of butyrate by the genetically engineered bacteria are provided in Table 7.

TABLE 7 Exemplary Polypeptide Sequences for Butyrate Production Description Sequence Bcd2 MDLNSKKYQMLKELYVSFAENEVKPLATELDEEERF SEQ ID NO: 62 PYETVEKMAKAGMMGIPYPKEYGGEGGDTVGYIMAV EELSRVCGTTGVILSAHTSLGSWPIYQYGNEEQKQK FLRPLASGEKLGAFGLTEPNAGTDASGQQTTAVLDG DEYILNGSKIFITNAIAGDIYVVMAMTDKSKGNKGI SAFIVEKGTPGFSFGVKEKKMGIRGSATSELIFEDC RIPKENLLGKEGQGFKIAMSTLDGGRIGIAAQALGL AQGALDETVKYVKERVQFGRPLSKFQNTQFQLADME VKVQAARHLVYQAAINKDLGKPYGVEAAMAKLFAAE TAMEVTTKAVQLHGGYGYTRDYPVERMMRDAKITEI YEGTSEVQRMVISGKLLK etfB3 MNIVVCIKQVPDTTEVKLDPNTGTLIRDGVPSIINP SEQ ID NO: 63 DDKAGLEEAIKLKEEMGAHVTVITMGPPQADMALKE ALAMGADRGILLTDRAFAGADTWATSSALAGALKNI DFDIIIAGRQAIDGDTAQVGPQIAEHLNLPSITYAE EIKTEGEYVLVKRQFEDCCHDLKVKMPCLITTLKDM NTPRYMKVGRIYDAFENDVVETWTVKDIEVDPSNLG LKGSPTSVFKSFTKSVKPAGTIYNEDAKTSAGIIID KLKEKYII etfA3 MGNVLVVIEQRENVIQTVSLELLGKATEIAKDYDTK SEQ ID NO: 64 VSALLLGSKVEGLIDTLAHYGADEVIVVDDEALAVY TTEPYTKAAYEAIKAADPIVVLFGATSIGRDLAPRV SARIHTGLTADCTGLAVAEDTKLLLMTRPAFGGNIM ATIVCKDFRPQMSTVRPGVMKKNEPDETKEAVINRF KVEFNDADKLVQVVQVIKEAKKQVKIEDAKILVSAG RGMGGKENLDILYELAEIIGGEVSGSRATIDAGWLD KARQVGQTGKTVRPDLYIACGISGAIQHIAGMEDAE FIVAINKNPEAPIFKYADVGIVGDVHKVLPELISQL SVAKEKGEVLAN Ter MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRIT SEQ ID NO: 65 AEVKAGAKAPKNVLVLGCSNGYGLASRITAAFGYGA ATIGVSFEKAGSETKYGTPGWYNNLAFDEAAKREGL YSVTIDGDAFSDEIKAQVIEEAKKKGIKFDLIVYSL ASPVRTDPDTGIMHKSVLKPFGKTFTGKTVDPFTGE LKEISAEPANDEEAAATVKVMGGEDWERWIKQLSKE GLLEEGCITLAYSYIGPEATQALYRKGTIGKAKEHL EATAHRLNKENPSIRAFVSVNKGLVTRASAVIPVIP LYLASLFKVMKEKGNHEGCIEQITRLYAERLYRKDG TIPVDEENRIRIDDWELEEDVQKAVSALMEKVTGEN AESLTDLAGYRHDFLASNGFDVEGINYEAEVERFDR I ThiA MREVVIASAARTAVGSFGGAFKSVSAVELGVTAAKE SEQ ID NO: 66 AIKRANITPDMIDESLLGGVLTAGLGQNIARQIALG AGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDAD IMLVGGAENMSMSPYLVPSARYGARMGDAAFVDSMI KDGLSDIFNNYHMGITAENIAEQWNITREEQDELAL ASQNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDKD EYIKPGTTMEKLAKLRPAFKKDGTVTAGNASGINDG AAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKIM GYGPVPATKKALEAANMTIEDIDLVEANEAFAAQSV AVIRDLNIDMNKVNVNGGAIAIGHPIGCSGARILTT LLYEMKRRDAKTGLATLCIGGGMGTTLIVKR Hbd MKLAVIGSGTMGSGIVQTFASCGHDVCLKSRTQGAI SEQ ID NO: 67 DKCLALLDKNLTKLVTKGKMDEATKAEILSHVSSTT NYEDLKDMDLIIEASVEDMNIKKDVFKLLDELCKED TILATNTSSLSITEIASSTKRPDKVIGMHFFNPVPM MKLVEVISGQLTSKVTFDTVFELSKSINKVPVDVSE SPGFVVNRILIPMINEAVGIYADGVASKEEIDEAMK LGANHPMGPLALGDLIGLDVVLAIMNVLYTEFGDTK YRPHPLLAKMVRANQLGRKTKIGFYDYNK Crt2 MSTSDVKVYENVAVEVDGNICTVKMNRPKALNAINS SEQ ID NO: 68 KTLEELYEVFVDINNDETIDVVILTGEGKAFVAGAD IAYMKDLDAVAAKDFSILGAKAFGEIENSKKVVIAA VNGFALGGGCELAMACDIRIASAKAKFGQPEVTLGI TPGYGGTQRLTRLVGMAKAKELIFTGQVIKADEAEK IGLVNRVVEPDILIEEVEKLAKIIAKNAQLAVRYSK EAIQLGAQTDINTGIDIESNLFGLCFSTKDQKEGMS AFVEKREANFIKG Pbt MRSFEEVIKFAKERGPKTISVACCQDKEVLMAVEMA SEQ ID NO: 69 RKEKIANAILVGDIEKTKEIAKSIDMDIENYELIDI KDLAEASLKSVELVSQGKADMVMKGLVDTSIILKAV LNKEVGLRTGNVLSHVAVFDVEGYDRLFFVTDAAMN LAPDTNIKKQIIENACTVAHSLDISEPKVAAICAKE KVNPKMKDTVEAKELEEMYERGEIKGCMVGGPFAID NAVSLEAAKHKGINHPVAGRADILLAPDIEGGNILY KALVFFSKSKNAGVIVGAKAPIILTSRADSEETKLN SIALGVLMAAKA Buk MSKIFKILTINPGSTSTKIAVEDNEDLVFEKTLRHS SEQ ID NO: 70 SEEIGKYEKVSDQFEFRKQVIEEALKEGGVKTSELD AVVGRGGLLKPIKGGTYSVSAAMIEDLKVGVLGEHA SNLGGIIAKQIGEEVNVPSYIVDPVVVDELEDVARI SGMPEISRASVVHALNQKAIARRYAREINKKYEDIN LIVAHMGGGVSVGAHKNGKIVDVANALDGEGPFSPE RSGGLPVGALVKMCFSGKYTQDEIKKKIKGNGGLVA YLNTNDAREVEERIEAGDEKAKLVYEAMAYQISKEI GASAAVLKGDVKAILLTGGIAYSKMFTEMIADRVKF IADVKVYPGEDEMIALAQGGLRVLTGEEEAQVYDN TesB MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFG SEQ ID NO: 41 GQVVGQALYAAKETVPEERLVHSFHSYFLRPGDSKK PIIYDVETLRDGNSFSARRVAAIQNGKPIFYMTASF QAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLL PPVLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQV WIRANGSVPDDLRVHQYLLGYASDLNFLPVALQPHG IGFLEPGIQIATIDHSMWFHRPFNLNEWLLYSVEST SASSARGFVRGEFYTQDGVLVASTVEGVMRNHN*

The gene products of the bcd2, etfA3, and etfB3 genes in Clostridium difficile form a complex that converts crotonyl-CoA to butyryl-CoA, which may function as an oxygen-dependent co-oxidant. In some embodiments, because the genetically engineered bacteria of the invention are designed to produce butyrate in a microaerobic or oxygen-limited environment, e.g., the mammalian gut, oxygen dependence could have a negative effect on butyrate production in the gut. It has been shown that a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) can functionally replace this three-gene complex in an oxygen-independent manner. In some embodiments, the genetically engineered bacteria comprise a ter gene, e.g., from Treponema denticola, which can functionally replace all three of the bcd2, etfB3, and etfA3 genes, e.g., from Peptoclostridium difficile. In this embodiment, the genetically engineered bacteria comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose..

In some embodiments, the genetically engineered bacteria of the invention comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile; ter, e.g., from Treponema denticola; one or more of bcd2, etfB3, and effA3, e.g., from Peptoclostridium difficile; and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

The gene products of pbt and buk convert butyrylCoA to Butyrate. In some embodiments, the pbt and buk genes can be replaced by a tesB gene. tesB can be used to cleave off the CoA from butyryl-coA. In one embodiment, the genetically engineered bacteria comprise bcd2, etfB3, effA3, thiA1, hbd, and crt2, e.g., from Peptoclostridium difficile, and tesB from E. Coli and produce butyrate in low-oxygen conditions, in the presence of molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In one embodiment, the genetically engineered bacteria comprise ter gene (encoding trans-2-enoynl-CoA reductase) e.g., from Treponema denticola, thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and tesB from E. Coli , and produce butyrate in low-oxygen conditions,in the presence of specific molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with hunger, appetite, craving, obesity, metablic syndrome, insulin resistance, liver damage, or other condition(s) such as inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells. In some embodiments, the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis.

In some embodiments, the local production of butyrate protects against diet-induced obesity (Lin et al., 2012). In some embodiments, the local production of butyrate protects against diet-induced obesity without causing decreased food intake (Lin et al., 2012). In some embodiments, local butyrate production reduces gut inflammation, a symptom of metabolic disease.

In one embodiment, the bcd2 gene has at least about 80% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene has at least about 85% identity with SEQ ID NO: 53. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 53. In one embodiment, the bcd2 gene has at least about 95% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. Accordingly, in one embodiment, the bcd2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene comprises the sequence of SEQ ID NO: 53. In yet another embodiment the bcd2 gene consists of the sequence of SEQ ID NO: 53.

In one embodiment, the etfB3 gene has at least about 80% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 54. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 54. In one embodiment, the e03 gene has at least about 95% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. Accordingly, in one embodiment, the e033 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene comprises the sequence of SEQ ID NO: 54. In yet another embodiment the etfB3 gene consists of the sequence of SEQ ID NO: 54.

In one embodiment, the etfA3 gene has at least about 80% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. Accordingly, in one embodiment, the e03 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 55. In yet another embodiment the etfA3 gene consists of the sequence of SEQ ID NO: 55.

In one embodiment, the thiA1 gene has at least about 80% identity with SEQ ID NO: 56. In another embodiment, the thiA1 gene has at least about 85% identity with SEQ ID NO: 56. In one embodiment, the thiA1 gene has at least about 90% identity with SEQ ID NO: 56. In one embodiment, the thiA1 gene has at least about 95% identity with SEQ ID NO: 56. In another embodiment, the thiAl gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. Accordingly, in one embodiment, the thiA1 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. In another embodiment, the thiAl gene comprises the sequence of SEQ ID NO: 56. In yet another embodiment the thiAl gene consists of the sequence of SEQ ID NO: 56.

In one embodiment, the hbd gene has at least about 80% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 57. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 57. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. Accordingly, in one embodiment, the hbd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 57. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 57.

In one embodiment, the crt2 gene has at least about 80% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. Accordingly, in one embodiment, the crt2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 58. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 58.

In one embodiment, the pbt gene has at least about 80% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 59. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 59. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. Accordingly, in one embodiment, the pbt gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 59. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 59.

In one embodiment, the buk gene has at least about 80% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 60. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 60. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. Accordingly, in one embodiment, the buk gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 60. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 60.

In one embodiment, the ter gene has at least about 80% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 61. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 61. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. Accordingly, in one embodiment, the ter gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 61. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 61.

In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 15.

In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. Accordingly, in one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In yet another embodiment one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.

In some embodiments, one or more of the butyrate biosynthesis genes is a synthetic butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Treponema denticola butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a C. glutamicum butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Peptoclostridicum difficile butyrate biosynthesis gene. The butyrate gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.

In some embodiments, the genetically engineered bacteria comprise a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing butyrate. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production. In some embodiments, the local production of butyrate reduces food intake and ameliorates metabolic disease (Lin et al., 2012). In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In one embodiment, the butyrate gene cassette is directly operably linked to a first promoter. In another embodiment, the butyrate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the butyrate gene cassette is operably linked to a promoter that it is not naturally linked to in nature.

In some embodiments, the butyrate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the butyrate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the butyrate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.

The butyrate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the butyrate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the butyrate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located on a plasmid in the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.

In some embodiments, the butyrate gene cassette is expressed on a low- copy plasmid. In some embodiments, the butyrate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of butyrate.

Acetate

In some embodiments, the genetically engineered bacteria of the invention comprise an acetate gene cassette and produce acetate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of acetate biosynthesis genes. Unmodified bacteria comprising acetate biosynthesis genes are known in the art and are capable of consuming various substrates to produce acetate under aerobic and/or anaerobic conditions (see, e.g., Ragsdale et al., 2008). In some embodiments, the genetically engineered bacteria of the invention comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced. In some embodiments, the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from Escherichia coli. In some embodiments, the genetically engineered bacteria comprise anaerobic acetate biosynthesis genes, e.g., from Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and/or Thermoacetogenium. The genetically engineered bacteria may comprise genes for aerobic acetate biosynthesis or genes for anaerobic or microaerobic acetate biosynthesis. In some embodiments, the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes. In some embodiments, the genetically engineered bacteria comprise a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing acetate. In some embodiments, one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production. In some embodiments, the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid.

In some embodiments, the genetically engineered bacteria produce acetate and butyrate, as described herein (see, e.g., FIG. 13 and FIG. 14). GLP-1

In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-1 or proglucagon. GLP-1 and several other insulin and satiety regulating peptides result from cleaved of preproglucagon. Preproglucagon is proteolytically cleaved in a tissue-specific manner. Post-translational processing in the gut and brain by prohormone convertases results in the secretion of GLP-1 and GLP-2, while the glucagon sequence remains in a larger peptide, glicentin or glicentin-related pancreatic peptide (GRPP) and oxyntomodulin. Glucagon-like peptide 1 (GLP-1) is produced by intestinal cells, e.g., ileal L cells, and is capable of stimulating insulin secretion and the differentiation of insulin-secreting cells and inhibiting glucagon secretion. GLP-1 is capable of restoring glucose sensitivity and increasing satiety.

Glucagon-like peptide 1 (GLP-1) is also used to treat those suffering from non-alcoholic steatohepatitis by reducing the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition. Glucagon-like peptide 1 is well known to those of skill in the art. For example, glucagon-like peptide 1 has been used to stimulate insulin secretion in the treatment of type-two diabetes and non-alcoholic steatohepatitis (NASH). See, for example, Armstrong, et al., J. of Hepatology, 64:399-408 (2016); Bernsmeier, et al., PLOS One, 9(1): e87488 (2014); Kjems, et al., Diabetes, 52:380-386 (2003); Knudsen et al., J. Med. Chem., 43:1664-1669 (2000); MacDonald, et al., Diabetes, 51(supp. 3):5434-5442 (2002); Werner, et al., Regulatory Peptides, 164:58-34 (2010); Drucker and Nauck, Lancet, 368:1696-1705 (2006); Jiminez-Solem, et al., Cur. Opinion in Mol. Therap., 12(6):760-797 (2010); Schnabel, et al., Vasc. Health and Risk Mgmt., 2(1):69-77 (2006); and WO1995/017510, the entire contents of each of which are expressly incorporated herein by reference.

Proteolytic cleavage of proglucagon produces GLP-1 and GLP-2. GLP-1 adminstration has therapeutic potential in treating type 2 diabetes (Gallwitz et al., 2000). The genetically engineered bacteria may comprise any suitable gene encoding GLP-1 or proglucagon, e.g., human GLP-1 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-1 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-1 analog (see, e.g., Gallwitz et al., 2000). In some embodiments, the gene encoding GLP-1 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-1 production, and/or increase metabolic disease attenuation potency. In some embodiments, the local production of GLP-1 induces insulin secretion and/or differentiation of insulin-secreting cells. In some embodiments, the local production of GLP-1 produces satiety in a subject and ameliorates obesity. In some embodiments, the genetically engineered bacteria are capable of expressing GLP-1 or proglucagon in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

TABLE 8 GLP-1 Polynucleotide Sequences Description Sequence GLP-1 (1-37), with ATGGACGAGTTCGAACGCCACG initiation met codon; CGGAGGGAACTTTCACTTCTGA codon optimized for TGTTTCTAGCTATTTGGAGGGC expression in E. coli. CAGGCTGCGAAAGAGTTTATTG SEQ ID NO: 71 CTTGGCTGGTTAAAGGTCGTGG TTAA GLP1 (1-37) codon GACGAGTTCGAACGCCACGCGG optimized for expression AGGGAACTTTCACTTCTGATGT in E. coli. TTCTAGCTATTTGGAGGGCCAG SEQ ID NO: 72 GCTGCGAAAGAGTTTATTGCTT GGCTGGTTAAAGGTCGTGGTTA A

TABLE 9 GLP-1 Polypeptide Sequences Description Sequence GLP-1 (1-37) HDEFERHAEGTFTSDVSSYLEGQAAKEFIAW SEQ ID NO: 73 LVKGRG GLP-1 (1-37) H→M substitution MDEFERHAEGTFTSDVSSYLEGQAAKEFIAW SEQ ID NO: 74 LVKGRG GLP-1-(7-37) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG SEQ ID NO: 75 GLP-1-(7-36)NH2 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR SEQ ID NO: 76 glucagon preproprotein MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSR (NP_002045.1) 1-20 is signal SFSASQADPLSDPDQMNEDKRHSQGTFTSDY peptide SKYLDSRRAQDFVQWLMNTKRNRNNIAKRHD SEQ ID NO: 77 EFERHAEGTFTSDVSSYLEGQAAKEFIAWLV KGRGRRDFPEEVAIVEELGRRHADGSFSDEM NTILDNLAARDFINWLIQTKITDRK Proglucagon (Signal peptide 1- RSLQDTEEKSRSFSASQADPLSDPDQMNEDK 20; Giucagon-like. peptide 1 RHSQGTFTSDYSKYLDSRRAQDFVQWLMNTK (92-128); Glucagon-like peptide RNRNNIAKRHDEFERHAEGTFTSDVSSYLEG 2 146-178 QAAKEFIAWLVKGRGRRDFPEEVAIVEELGR SEQ ID NO: 78 RHADGSFSDEMNTILDNLAARDFINWLIQTK ITDRK Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEQ ID NO: 79 Glicentin RSLQDTEEKSRSFSASQADPLSDPDQMNEDK SEQ ID NO: 80 RHSQGTFTSDYSKYLDSRRAQDFVQWLMNTK RNRNNIA Glicentin related peptide RSLQDTEEKSRSFSASQADPLSDPDQMNED SEQ ID NO: 81 Oxyntomodulin HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKR SEQ ID NO: 82 NRNNIA

The circulating active form of GLP-1 is GLP-1(7-37), which has a very short biological half-life of the order of just a few minutes in blood. The relatively low stability of GLP-1 (3-5 min) has significantly limited its clinical utility because of the rapid degradation catalyzed by the enzyme dipeptidyl peptidase IV (DPP-IV), but also other enzymes such as neutral endopeptidase (NEP), plasma kallikrein or plastnin. One strategy to prolong in vivo half-life is stabilization towards degradation by DPPIV, which preferably cleaves N-terminal Xaa-Pro or Xaa-Ala dipeptide sequences. Alteration of that N-terminal sequence, especially the second amino acid, has proven to reduce degradation by DPPIV (e.g., reviewed in Lorenz et al., Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity; Bioorganic & Medicinal Chemistry Letters, 23 (14); 4011-4018). In some embodiments, the genetically engineered bacteria comprise a cassette encoding GLP-1 fragment or variant, in which the DPP-IV is mutated, such that it can no longer be cleaved by the enzyme.

GLP-1 is released in a tissue specific manner, though post-translational processing of pre-pro-glucagon, from the neuroendocrine L-cells predominantly in two forms, GLP-1 (7-36) amide, which constitutes approximately 80% of circulating GLP-1, and GLP-1 (7-37) amide. GLP-1 (1-36 amide) is predominantly secreted in the pancreas, whereas GLP-1 (1-37) is secreted in the ileum and hypothalamus.

In addition, full length GLP-1-(1-37) is produced in much smaller amounts. This full-length form of GLP-1(1-37), was previously thought to be inactive, but was found to stimulate rat intestinal epithelial cells to become glucose-responsive insulin-secreting cells, i.e., full length GLP-1 could convert intestinal epithelial progenitors in the small intestine into insulin-producing cells (Suzuki et al., Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells into insulin-producing cells; Proc Natl Acad Sci U S A. 2003 Apr. 29; 100(9): 5034-5039). While the amounts of GLP-1 (1-37) produced endogenously likely are not sufficient for these effects, secretion of large amounts of GLP-1, e.g., by the genetically engineered bacteria, are likely sufficient to alter a balance in the developmental environment of the intestinal epithelia, leading to the induction of insulin-producing cells from intestinal epithelial progenitors. As such, secretion of full-length GLP-1 by the genetically engineered bacteria of the disclosure is a novel therapeutic strategy for the treatment of a number of diseases related to dysregulation of insulin production and/or secretion, including diabetes.

GLP-1 analogs, which exhibit extended stability in serum, have become important in the clinic. Exendin-4, a peptide produced in the salivary glands of the Gila monster (Heloderma suspectum), possesses similar glucose regulatory function to the human GLP-1 peptide. In exendin-4, the second amino acid is a Gly rendering it resistant to DPPIV mediated degradation. Furthermore, the Leu21-Ser39 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure, leading to enhanced helicity and stability of the peptide (see Lorenz et al. for review). Exenatide BID is a synthetic version of exendin-4, represents the first GLP-1 RA approved in 2005 as antidiabetic therapy for the treatment of T2DM. Following the FDA approval of exendin-4, liraglutide and albiglutide, which are long-acting GLP-1 analogs using palmitic acid conjugation and albumin fusion, respectively, were approved. Many other strategies have also been employed to achieve long-acting activity of GLP-1, including dimerization, intra-molecular conjugation, and additional variant positive charged amino acids on the N terminus. Table 10 lists non-limiting examples of GLP-1R agonists. In some embodiments, the genetically engineered bacteria comprise a gene encoding Exenatide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Liraglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Lixisenatide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Albiglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Dulaglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Taspoglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Semaglutide.

TABLE 10 Non-limiting examples of GLP-1R agonists Name and SEQ ID NO Sequence Short description Exenatide HGEGTFTSDLSKQMEE Second amino acid is a Gly SEQ ID NO: 83 EAVRLFIEWLKNGGPS rendering it resistant to SGAPPPS DPPIV mediated degradation. Furthermore, the Leu21-Ser39 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure, leading to enhanced helicity and stability of the peptide Liraglutide HAEGTFTSDVSSYLEG a close structural homolog to SEQ ID NO: 84 QAAKEEFIIAWLVKGR GLP-1(7-37) with 97% G sequence identity to the native hormone. Lys in position 34 is substituted by Arg and a palmitic acid is conjugated to Lys in position 26 via a glutamate spacer Lixisenatide HGEGTFTSDLSKQMEE synthetic analog of exendin- SEQ ID NO: 85 EAVRLFIEWLKNGGPS 4. Compared to exendin-4, SGAPPSKKKKKK six Lys residues have been added to the C-terminus (also amidated), while one Pro in the C-terminal region has been deleted. Albiglutide HGEGTFTSDVSSYLEG two copies of GLP-1 are SEQ ID NO: 86 QAAKEFIAWLVKGRH fused as tandem repeat to the GEGTFTSDVSSYLEGQ N-terminus of albumin. AAKEFIAWLVKGRDA DPPIV-resistance is HKSEVAHRFKDLGEEN achieved by a single FKALVLIAFAQYLQQC substitution, Ala for Gly, at PFEDHVKLVNEVTEFA the DPPIV cleavage site. KTCVADESAENCDKSL HTLFGDKLCTVATLRE TYGEMADCCAKQEPE RNECFLQHKDDNPNLP RLVRPEVDVMCTAFH DNEETFLKKYLYEIAR RHPYFYAPELLFFAKR YKAAFTECCQAADKA ACLLPKLDELRDEGKA SSAKQRLKCASLQKFG ERAFKAWAVARLSQR FPKAEFAEVSKLVTDL TKVHTECCHGDLLECA DDRADLAKYICENQDS ISSKLKECCEKPLLEKS HCIAEVENDEMPADLP SLAADFVESKDVCKN YAEAKDVFLGMFLYE YARRHPDYSVVLLLRL AKTYETTLEKCCAAA DPHECYAKVFDEFKPL VEEPQNLIKQNCELFE QLGEYKFQNALLVRY TKKVPQVSTPTLVEVS RNLGKVGSKCCKHPE AKRMPCAEDYLSVVL NQLCVLHEKTPVSDRV TKCCTESLVNRRPCFS ALEVDETYVPKEFNAE TFTFHADICTLSEKERQ IKKQTALVELVKHKPK ATKEQLKAVMDDFAA FVEKCCKADDKETCFA EEGKKLVAASQAALG L Dulaglutide HGEGTFTSDVSSYLEE A recombinant fusion SEQ ID NO: 87 QAAKEFIAWLVKGGG protein, which consists of GGGGSGGGGSGGGGS two GLP-1 peptides AESKYGPPCPPCPAPE covalently linked by a small AAGGPSVFLFPPKPKD peptide [tetraglycyl-1- TLMISRTPEVTCVVVD seryltetraglycyl-1- VSQEDPEVQFNWYVD seryltetraglycyl-1-seryl-1- GVEVHNAKTKPREEQF alanyl] to a human IgG4-Fc NSTYRVVSVLTVLHQD heavy chain variant. WLNGKEYKCKVSNKG Compared to natural GLP-1, LPSSIEKTISKAKGQPR the GLP-1 moieties contain EPQVYTLPPSQEEMTK amino acid substitutions NQVSLTCLVKGFYPSD (Ala8→Gly, Gly26→Glu, IAVEWESNGQPENNYK Arg36→Gly) to ensure TTPPVLDSDGSFFLYSR protection from DPPIV LTVDKSRWQEGNVFS cleavage as well as CSVMHEALHNHYTQK maintenance of the potency SLSLSLG of the construct. Taspoglutide His-Aib-Glu-Gly-Thr- a close analog of natural SEQ ID NO: 88 Phe-Thr-Ser-Asp-Val-Ser- GLP-1(7-36) in which the Ser-Tyr-Leu-Gly-Gly- unnatural amino acid Gln-Ala-Ala-Lys-Glu- aminoisobutyric acid (Aib) Phe-Ile-Ala-Trp-Leu-Val- has been introduced in Lys-Aib-Arg-NH₂ position 8 and 35 in order to avoid degradation by DITIV, hut also by other serine proteases such as plasma kallikrein and plasmin. Semaglutide MAGAPGPLRLALLLLG SEQ ID NO: 89 MVGRAGPRPQGATVS LWETVQKWREYRRQC QRSLTEDPPPATDLFC NRTFDEYACWPDGEP GSFVNVSCPWYLPWA SSVPQGHVYRFCTAEG LWLQKDNSSLPWRDL SECEESKRGERSSPEEQ LLFLYIIYTVGYALSFS ALVIASAILLGFRHLHC TRNYIHLNLFASFILRA LSVFIKDAALKWMYST AAQQHQWDGLLSYQD SLSCRLVFLLMQYCVA ANYYWLLVEGVYLYT LLAFSVLSEQWIFRLY VSIGWGVPLLFVVPWG IVKYLYEDEGCWTRNS NMNYWLIIRLPILFAIG VNFLIFVRVICIVVSKL KANLMCKTDIKCRLA KSTLTLIPLLGTHEVIF AFVMDEHARGTLRFIK LFTELSFTSFQGLMVAI LYCFVNNEVQLEFRKS WERWRLEHLHIQRDSS MKPLKCPTSSLSSGAT AGSSMYTATCQASCS

In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 stimulates the rate of insulin secretion in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 inhibits and lowers plasma glucose produced in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the level of lipotoxic metabolites in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the degree of pro-inflammatory substrate in the body. In one embodiment, GLP-1 decreases the level of insulin resistance (IR) in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the level of hepatic lipid deposition in the body. Methods for measuring the insulin secretion rates and glucose levels are well known to one of ordinary skill in the art. For example, blood samples taken periodically, and standard statistical analysis methods may be used to determine the insulin secretion rates and plasma glucose levels in a subject.

GLP-1 and/or a GLP-1R agonist of Table 10 may be expressed or modified in bacteria of this disclosure in order to enhance insulin stimulation and reduce plasma glucose levels in subjects having liver disease, such as NASH. Specifically, when GLP-1 and/or a GLP-1R agonist of Table 10 is expressed in the engineered bacterial cells of the disclosure, the expressed GLP-1 and/or a GLP-1R agonist of Table 10 will reduce the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition in the subject.

GLP-1 and/or a GLP-1R agonist of Table 10 may be expressed or modified in bacteria of this disclosure in order to enhance insulin stimulation and reduce plasma glucose levels in subjects having type two diabetes, obesity, and/or metabolic syndrome, or metabolic syndrome related disorders, including cardiovascular disorders, and obesity in a subject.

In one embodiment, the bacterial cell comprises one or more genes encoding a GLP-1 and/or a GLP-1R agonist of Table 10. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a glucagon-like peptide 1 operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a glucagon-like peptide 1. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a glucagon-like peptide 1.

Multiple distinct embodiments of GLP-1 and/or a GLP-1R agonist of Table 10 are known in the art. In some embodiments, the glucagon-like peptide 1 is encoded by a gene derived from a bacterial species. In some embodiments, a glucagon-like peptide 1 is encoded by a gene derived from a non-bacterial species. In some embodiments, a glucagon-like peptide 1 is encoded by a gene derived from a eukaryotic species, e.g. homo sapiens. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed in an organism of the genus or species that includes, but is not limited to, Lactobacillus spp., such as Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus brevis, or Lactobacillus gasseri; Bifidobacterium spp., such as Bifidobacterium longum; Bacillus spp., such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus; and Streptomyces spp., such as Streptomyces lividans.

In one embodiment, the gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the gene encoding the glucagon-like peptide 1 has been codon-optimized for use in Escherichia coli. In another embodiment, the gene encoding the glucagon-like peptide 1 has been codon-optimized for use in Lactococcus. When the gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 is expressed in the engineered bacterial cells, the bacterial cells express more GLP-1 and/or a GLP-1R agonist of Table 10 than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous gene encoding a GLP-1 and/or a GLP-1R agonist of Table 10 may be used to express more GLP-1 and/or a GLP-1R agonist of Table 10 to treat liver disease, such as nonalcoholic steatohepatitis, type two diabetes, metabolic syndrome, and metabolic syndrome related disorders, including cardiovascular disorders and obesity in a subject.

Assays for testing the activity of a GLP-1 and/or a GLP-1R agonist of Table 10 or a glucagon-like peptide 1 receptor are well known to one of ordinary skill in the art. For example, glucose and insulin levels can be assessed by drawing plasma samples from subjects previously administered intravenous infusions of the glucagon- like peptide 1 as described in Kjems, et al., Diabetes, 52:380-386 (2003), the entire contents of which are expressly incorporated herein by reference. Briefly, plasma samples from a subject are treated with heparin and sodium fluoride, centrifuged, and plasma glucose levels measured by a glucose oxidase technique. Likewise, the plasma insulin concentrations are measured by a two-site insulin enzyme linked immunosorbent method. Alternatively, baby hamster kidney cells can be used to assay structure-activity relationships of glucagon-like peptide 1 derivatives (see, for example, Knudsen et al., J. Med. Chem., 43:1664-1669 (2000), the entire contents of which are expressly incorporated herein by reference). The present disclosure encompasses genes encoding a GLP-1 and/or a GLP-1R agonist of Table 10 comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein.

In some embodiments, the gene encoding a GLP-1 and/or a GLP-1R agonist of Table 10 is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

In one embodiment, the gene encoding the glucagon-like peptide 1 is from Homo sapiens. In one embodiment, the gene encoding the glucagon-like peptide 1 is from Lactobacillus spp. In one embodiment, the Lacotbacillus spp. is Lactobacillus plantarum WCFS1, Lactobacillus plantarum 80, Lactobacillus johnsonii NCC533, Lactobacillus johnsonii 100-100, Lactobacillus acidophilus NCFM ATCC700396, Lactobacillus brevis ATCC 367, Lactobacillus gasseri ATCC 33323, or Lactobacillus acidophilus. In another embodiment, the gene encoding the glucagon-like peptide 1 is from a Bifidobacterium spp. In one embodiment, the Bifidobacterium spp. is Bifidobacterium longum NCC2705, Bifidobacterium longum DJ010A, Bifidobacterium longum BB536, or Bifidobacterium longum SBT2928. In another embodiment, the gene encoding the glucagon-like peptide 1 is from Bacillus spp. In one embodiment, the Bacillus spp is Bacillus subtilis, or Bacillus licheniformis, or Bacillus lentus, or Bacillus brevis, or Bacillus stearothermophilus, or Bacillus alkalophilus, or Bacillus amyloliquefaciens, or Bacillus coagulans, or Bacillus circulans, or Bacillus lautus. In another embodiment, the gene encoding the glucagon-like peptide 1 is from Streptomyces spp. In one embodiment, the Streptomyces spp. is Streptomyces lividans. Other genes encoding glucagon-like peptide 1 are well-known to one of ordinary skill in the art and described in, for example, MacDonald, et al., Diabetes, 51(supp. 3):S434-S442 (2002) and WO1995/017510.

In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 80% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment, the gene encoding the glucagon-like peptide 1 has at least about 85% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 90% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 95% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment, the gene encoding the glucagon-like peptide 1 has at least about 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. Accordingly, in one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding SEQ ID NO:40. In another embodiment, the gene encoding the glucagon-like peptide 1 comprises a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In yet another embodiment the gene encoding the glucagon-like peptide 1 consists of a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72.

In one embodiment, the gene encoding the glucagon-like peptide 1 is directly operably linked to a first promoter. In another embodiment, the gene encoding the glucagon-like peptide 1 is indirectly operably linked to a first promoter. In one embodiment, the gene encoding the glucagon-like peptide 1 is operably linked to a promoter that it is not naturally linked to in nature.

In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed under the control of a constitutive promoter. In another embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the glucagon-like peptide 1 is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. Inducible promoters are described in more detail infra.

The gene encoding the glucagon-like peptide 1 may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located on a plasmid in the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located in the chromosome of the bacterial cell.

In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed on a low-copy plasmid. In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the glucagon-like peptide 1, thereby reducing the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition prevalent to those suffering from non-alcoholic steatohepatitis.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1 (1-37), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 73. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1 (1-37) H->M substitution), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 74. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1-(7-37), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 75. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1-(7-36), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 76.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding glucagon preproprotein (NP_002045.1), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Proglucagon, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 78. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glucagon, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 79. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glicentin), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 80 In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glicentin related peptide), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 81. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Oxyntomodulin. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 82.

In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. Accordingly, in one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. In yet another embodiment one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 73 through SEQ ID NO: 82.

In embodiments, the pro-glucagon derived polypeptides, GLP-1 polypeptides, GLP-1 analogs described herein, and functional variants or fragments thereof are secreted. In some embodiments, the genetically engineered bacteria comprise one or more cassettes encoding pro-glucagon derived polypeptides, GLP-1 polypeptides, GLP-1 analogs, and/or functional variants or fragments and a secretion gene cassette and/or mutations generating a leaky phenotype. In some embodiments, a flagellar type III secretion pathway is used to secrete pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. In some embodiments, a Type V Autotransporter Secretion System is used to secrete pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. In some embodiments, a Hemolysin-based Secretion System is used to secrete the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein.. In alternate embodiments, the genetically engineered bacteria expressing the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein further comprise a non-native single membrane-spanning secretion system. As described herein. In some embodiments, the engineered bacteria expressing the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. have one or more deleted or mutated membrane genes to generate a leaky phenotype as described herein.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Exenatide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 83.

In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Liraglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 84. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Lixisenatide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 85. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Albiglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 86. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Dulaglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 87. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Taspoglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding

SEQ ID NO: 88. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Semaglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 89.

In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 83 through SEQ ID NO: 89.

IL-22

In some embodiments, the genetically engineered bacteria are capable of producing IL-22. Interleukin 22 (IL-22) cytokine can be produced by dendritic cells, lymphoid tissue inducer-like cells, natural killer cells and expressed on adaptive lymphocytes. Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of 11-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.

As described by Wang et al, (Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes, Nature 514, 237-241 (9 Oct. 2014)) mice which are deficient in IL-22 receptor and are fed a high-fat diet have a propensity to the development of metabolic disorders. Moreover, Wang et al found that administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. These results indicate that IL-22 shows metabolic benefits, from positively affecting insulin sensitivity to the preservation of gut barreier integrity. IL-22 further affects endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues.

Bile Salts

Bile salts (also called conjugated bile acids) are cholesterol derivatives synthesized in the liver which comprise a steroid ring component conjugated with either taurine (taurocholic acid; TCA) or glycine (glycochenodeoxycholic acid; GCDCA). Bile salts act as signaling molecules to regulate systemic endocrine functions, including triglyceride, cholesterol, and glucose homeostasis (Houten et al., EMBO J., 25:1419-1425 (2006) and Watanabe et al., Nature, 439:484-489 (2006)). Specifically, bile acids trigger cellular farnesoid X receptor (FXR)- and G-protein coupled receptor (TGR4)-mediated host responses. Additionally, bile salts have been shown to facilitate lipid absorption and repress bacterial cell growth in the small intestine, thereby influencing both host metabolic pathways and the microflora present in the gut (Jones et al., PNAS, 105(36):13580-13585 (2008) and Ridlon et al., J. Lipid Research, 47(2):241-259 (2006)).

Bile salts are stored in the gallbladder and then subsequently released into the duodenum via the common bile duct. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules, a process referred to as deconjugation, to produce the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non-reabsorbed bile acids enter the large intestine. Once in the large intestine, bile acids are amenable to further modification by microbial 7α-dehydroxylase enzymes to yield secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA) (Joyce et al., Gut Microbes, 5(5):669-674 (2014); Bhowmik et al., Accepted Article, doi:10.1002/prot.24971 (2015)).

It has been shown that bile salt metabolism is involved in host physiology (Ridlon et al., Current Opinion Gastroenterol., 30(3):332 (2014) and Jones et al., 2008). For example, it is known that the expression of bile salt hydrolase enzymes functionally regulates host lipid metabolism and play a role in cholesterol metabolism and transport, circadian rhythm, gut homeostasis/barrier function, weight gain, adiposity, and possibly gastrointestinal cancers in the host (Joyce et al., PNAS, 111(20):7421-7426 (2014); Zhou and Hylemon, Steroids, 86:62-68, (2014); Mitchell et al., Expert Opinion Biolog. Therapy, 13(5):631-642 (2013); and WO14/198857, the entire contents of each of which are expressly incorporated herein by reference). Specifically, potential effects of bile salt hydrolase-expressing bacteria on cholesterol metabolic pathways have been shown to upregulate the ATP binding cassette A1 (ABCA1), the ATP binding cassette G1 (ABCG1), the ATP binding cassette G5/G8 (ABCGS/G8), cholesterol 7 alpha-hydroxylase (CYP7A1), and liver X receptor (LXR), and to downregulate farnesoid X receptor (FXR), Niemann-Pick C1-like 1 (NPC1L1), and small heterodimer partner (SHP), which impacts cholesterol efflux, plasma HDL-C levels, biliary excretion, cholesterol catabolism, bile acid synthesis, cholesterol levels, and decreased intestinal cholesterol absorption, among other effects (Mitchel et al. (2014) and Zhou and Hylemon (2014)). Additionally, bile salt hydrolase activity has been shown to impact bile detoxification, gastrointestinal persistence, nutrition, membrane alterations, altered digestive functions (lipid malabsorption, weight loss), cholesterol lowering, cancer, and formation of gallstones (see Begley et al., Applied and Environmental Microbiology, 72(3):1729-1738 (2006)). Moreover, a Clostridium scindens bacterium expressing a 7α-dehydroxylase enzyme has been shown to produce resistance to C. difficile infection in hosts (Buffie et al., Nature, 517:205-208 (2015), and bile salt metabolism has been shown to play a role in both regulating the microbiome as well as in cirrhosis (Ridlon et al., Gut Microbes, 4(5):382-387 (2013) and Kakiyama et al., J. Hepatol., 58(5):949-955 (2013)). Thus, a need exists for treatments which address the metabolism of bile salts in subjects in order to treat and prevent diseases and disorders in which bile salts play a role, such as cardiovascular disease, metabolic disease, cirrhosis, gastrointestinal cancer, and C. difficile infection.

As used herein, the term “bile salt” or “conjugated bile acid” refers to a cholesterol derivative that is synthesized in the liver and consists of a steroid ring component that is conjugated with either glycine (glycochenodeoxycholic acid; GCDCA) or taurine (taurocholic acid; TCA). Bile salts are stored in the gallbladder and then subsequently released into the duodenum. Bile salts act as signaling molecules to regulate systemic endocrine functions including triglyceride, cholesterol, and glucose homeostasis, and also facilitate lipid absorption. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules to produce bile acids.

As used herein, the term “bile acid” or “unconjugated bile acid” refers to cholic acid (CA) or chenodeoxycholic acid (CDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non-reabsorbed bile acids enter the large intestine. In the large intestine, bile acids are amenable to further modification by microbial 7α-dehydroxylase enzymes to yield secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA). As used herein, the term “catabolism” refers to the processing, breakdown and/or degradation of a metabolite or a complex molecule, such as tryptophan or a bile salt, into compounds that are non-toxic or which can be utilized by the bacterial cell or can be exported inot the extracellular environment, where these compounds may function as effectors.

In one embodiment, the term “bile salt catabolism” refers to the processing, breakdown, and/or degradation of bile salts into unconjugated bile acid(s). In one embodiment, “abnormal catabolism” refers to any condition(s), disorder(s), disease(s), predisposition(s), and/or genetic mutations(s) that result in increased levels of bile salts. In one embodiment, “abnormal catabolism” refers to an inability and/or decreased capacity of a cell, organ, and/or system to process, degrade, and/or secrete bile salts. In healthy adult humans, 600 mg of bile salts are secreted daily. In one embodiment, said inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts is caused by the decreased endogenous deconjugation of bile salts, e.g., decreased endogenous deconjugation of bile salts into bile acids by the intestinal microbiota in the gut. In one embodiment, the inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts results from a decrease in the number of or activity of intestinal bile salt hydrolase (BSH)-producing microorganisms.

In one embodiment, a “disease associated with bile salts” or a “disorder associated with bile salts” is a disease or disorder involving the abnormal, e.g., increased, levels of bile salts in a subject. Alternatively, a disease or disorder associated with bile salts is a disease or disorder wherein a subject exhibits normal levels of bile salts, but wherein the subject would benefit from decreased levels of bile salts. Bile salts function to solubilize dietary fat and enable its absorption into host circulation, and healthy adult humans secrete about 600 mg of bile salts daily through the stool. Thus, decreasing increased levels of bile salts, or normal levels of bile salts, in a subject would result in less uptake of dietary fat, causing the subject's liver to pull cholesterol from systemic circulation as it attempts to synthesize more. Thus, in one embodiment, a subject having a disease or disorder associated with bile salts secretes about 600 mg of bile salts in their stool daily. In another embodiment, a subject having a disease or disorder associated with bile salts secretes more than 600 mg, 700 mg, 800 mg, 900 mg, or 1 g of bile salts in their stool daily.In one embodiment, a disease or disorder associated with bile salts is a cardiovascular disease. In another embodiment, a disease or disorder associated with bile salts is a metabolic disease. In another embodiment, a disease or disorder associated with bile salts is a liver disease, such as cirrhosis, nonalcoholic steatohepatitis (NASH), or progressive familialintrahepatic cholestasis type 2 (PFIC2).

As used herein, the terms “cardiovascular disease” or “cardiovascular disorder” are terms used to classify numerous conditions affecting the heart, heart valves, and vasculature (e.g., veins and arteries) of the body, and encompasses diseases and conditions including, but not limited to hypercholesterolemia, diabetic dyslipidemia, hypertension, arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, congestive heart failure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm, pulmonary embolism, primary hypertension, atrial fibrillation, stroke, transient ischemic attack, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque, acute coronary syndrome, acute ischemic attack, sudden cardiac death, peripheral vascular disease, coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease. As used herein, a subject having “hypercholesterolemia” may have a total cholesterol of greater than 4 mmol/L, and a low-density lipoprotein cholesterol (LDL) of greater than 3mmol/L.

As used herein, the term “bile salt hydrolase” enzyme refers to an enzyme involved in the cleavage of the amino acid sidechain of glycol- or tauro-conjugated bile acids to generate unconjugated bile acids (FIG. 2). Bile salt hydrolase (BSH) enzymes are well known to those of skill in the art. For example, bile salt hydrolase activity has been detected in Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Clostridum spp., Bacteroides spp., Methanobrevibacter spp., and Listeria spp. See, for example, Begley et al., Applied and Environmental Microbiology, 72(3):1729-1738 (2006); Jones et al., Proc. Natl. Acad. Sci., 105(36):13580-13585 (2008); Ridlon et al., J. Lipid Res., 47(2):241-259 (2006); and WO2014/198857, the entire contents of each of which are expressly incorporated herein by reference.

Bile Salt Hydrolases

The bacterial cells described herein comprise a heterologous gene encoding a bile salt hydrolase enzyme and are capable of deconjugating bile salts into unconjugated bile acids (see FIG. 27 and FIG. 28).

In one embodiment, the bile salt hydrolase enzyme increases the rate of bile salt catabolism in the cell. In one embodiment, the bile salt hydrolase enzyme decreases the level of bile salts in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of taurocholic acid (TCA) in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of glycochenodeoxycholic acid (GCDCA) in the cell or in the subject. Methods for measuring the rate of bile salt catabolism and the level of bile salts and bile acids are well known to one of ordinary skill in the art. For example, bile salts and acids may be extracted from a sample, and standard LC/MS methods may be used to determine the rate of bile salt catabolism and/or level of bile salts and bile acids.

In another embodiment, the bile salt hydrolase enzyme increases the level of bile acids in the cell or in the subject as compared to the level of bile salts in the cell or in the subject. In another embodiment, the bile salt hydrolase enzyme increases the level of cholic acid (CA) in the cell. In another embodiment, the bile salt hydrolase enzyme increases the level of chenodeoxycholic acid (CDCA) in the cell.

Enzymes involved in the catabolism of bile salts may be expressed or modified in the bacteria of the disclosure in order to enhance catabolism of bile salts. Specifically, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts into unconjugated bile acids when the bile salt hydrolase enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts, such as TCA or GCDCA, into CA and CDCA when the bile salt hydrolase enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme can catabolize bile salts to treat disorders associated with bile salts, including cardiovascular diseases, metabolic diseases, liver disease, such as cirrhosis or NASH, gastrointestinal cancers, and C. difficile infection.

In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a gene encoding a bile salt hydrolase enzyme from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a bile salt hydrolase enzyme. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a bile salt hydrolase enzyme, as well as at least one copy of a gene encoding a bile salt hydrolase enzyme from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a bile salt hydrolase enzyme. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a bile salt hydrolase enzyme.

Multiple distinct bile salt hydrolase enzymes are known in the art. In some embodiments, bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a non-bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene derived from a eukaryotic species, e.g., fungi. In one embodiment, the gene encoding the bile salt hydrolase enzyme is derived from an organism of the genus or species that includes, but is not limited to, Lactobacillus spp., such as Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus brevis, or Lactobacillus gasseri; Bifidobacterium spp., such as Bifidobacterium longum, Bifidobacterium bifidum, or Bifidobacterium adolescentis; Bacteroides spp., such as Bacteroides fragilis or Bacteroides vlugatus; Clostridium spp., such as Clostridium perfringens; Listeria spp., such as Listeria monocytogenes, Enterococcus spp., such as Enterococcus faecium or Enterococcus faecalis; Brucella spp., such as Brucella abortus; Methanobrevibacter spp., such as Methanobrevibacter smithii, Staphylococcus spp., such as Staphylococcus aureus, Mycobacterium spp., such as Mycobacterium tuberculosis; Salmonella spp., such as Salmonella enterica; Listeria spp., such as Listeria monocytogenes.

In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in the recombinant bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in Escherichia coli. In another embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in Lactococcus. When the gene encoding the bile salt hydrolase enzyme is expressed in the recombinant bacterial cells, the bacterial cells catabolize more bile salt than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme may be used to catabolize excess bile salts to treat a disorder associated with bile salts, such as cardiovascular disease, metabolic disease, liver disease, such as cirrhosis or NASH.

The present disclosure further comprises genes encoding functional fragments of a bile salt hydrolase enzyme or functional variants of a bile salt hydrolase enzyme. As used herein, the term “functional fragment thereof” or “functional variant thereof” of a bile salt hydrolase enzyme relates to an element having qualitative biological activity in common with the wild-type bile salt hydrolase enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated bile salt hydrolase enzyme is one which retains essentially the same ability to catabolize bile salts as the bile salt hydrolase enzyme from which the functional fragment or functional variant was derived. For example, a polypeptide having bile salt hydrolase enzyme activity may be truncated at the N-terminus or C-terminus and the retention of bile salt hydrolase enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional variant. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional fragment.

Assays for testing the activity of a bile salt hydrolase enzyme, a bile salt hydrolase enzyme functional variant, or a bile salt hydrolase enzyme functional fragment are well known to one of ordinary skill in the art. For example, bile salt catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in a recombinant bacterial cell that lacks endogenous bile salt hydrolase enzyme activity. Bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and Feighner, Applied Environ. Microbiol., 55:11-16 (1989) and Christiaens et al., Appl. Environ. Microbiol., 58:3792-3798 (1992), the entire contents of each of which are expressly incorporated herein by reference. Briefly, bacterial cultures that are grown overnight can be spotted onto LB bile agar supplemented with either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile. BSH activity can be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al., PNAS, 105(36):13580-13585 (2008), the entire contents of which are expressly incorporated herein by reference). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al., Gut Microbes, 3(3):186-202 (2012), the entire contents of which are expressly incorporated herein by reference. Alternatively, a mouse model can be used to assay bile salt and bile acid signatures in vivo (see, for example, Joyce et al., PNAS, 111(20):7421-7426 (2014), the entire contents of which are expressly incorporated herein by reference). The present disclosure encompasses genes encoding a bile salt hydrolase enzyme comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein.

In some embodiments, the gene encoding a bile salt hydrolase enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the bile salt hydrolase enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

In one embodiment, the gene encoding the bile salt hydrolase enzyme is from Lactobacillus spp. In one embodiment, the Lacotbacillus spp. is Lactobacillus plantarum WCFS1, Lactobacillus plantarum 80, Lactobacillus johnsonii NCC533, Lactobacillus johnsonii 100-100, Lactobacillus acidophilus NCFM ATCC700396, Lactobacillus brevis ATCC 367, Lactobacillus gasseri ATCC 33323, or Lactobacillus acidophilus. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from a Bifidobacterium spp. In one embodiment, the Bifidobacterium spp. is Bifidobacterium longum NCC2705, Bifidobacterium longum DJO10A, Bifidobacterium longum BB536, Bifidobacterium longum SBT2928, Bifidobacterium bifidum ATCC 11863, or Bifidobacterium adolescentis. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Bacteroides spp. In one embodiment, the Bacteroides spp. is Bacteroides fragilis or Bacteroides vlugatus. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Clostridium spp. In one embodiment, the Clostridum spp. is Clostridum perfringens MCV 185 or Clostridum perfringens 13. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Listeria spp. In one embodiment, the Listeria spp. is Listeria monocytogenes. In one embodiment, the gene encoding the bile salt hydrolase enzyme is from Methanobrevibacter spp. In one embodiment, the Methanobrevibacter spp. is Methanobrevibacter smithii. Other genes encoding bile salt hydrolase enzymes are well-known to one of ordinary skill in the art and described in, for example, Jones et al., PNAS, 105(36):13580-13585 (2008) and WO2014/198857. Table 11A lists non-limiting examples of bile salt hydrolases.

TABLE 11A Bile Salt Hydrolases Gene or Operon Sequence Bile salt hydrolase ATGTGTACTGCCATAACTTATCAATCTTATAATAATTACTTC from Lactobacillus GGTAGAAATTTCGATTATGAAATTTCATACAATGAAATGGTT plantarum ACGATTACGCCTAGAAAATATCCACTAGTATTTCGTAAGGTG SEQ ID NO: 90 GAGAACTTAGATCACCATTATGCAATAATTGGAATTACTGCT GATGTAGAAAGCTATCCACTTTACTACGATGCGATGAATGAA AAAGGCTTGTGTATTGCGGGATTAAATTTTGCAGGTTATGCT GATTATAAAAAATATGATGCTGATAAAGTTAATATCACACCA TTTGAATTAATTCCTTGGTTATTGGGACAATTTTCAAGTGTT AGAGAAGTGAAAAAGAACATACAAAAACTAAACTTGGTTAAT ATTAATTITAGTGAACAATTACCATTATCACCGCTACATTGG TTGGTTGCTGATAAACAGGAATCGATAGTTATTGAAAGTGIC AAAGAAGGACTAAAAATTTACGACAATCCAGTAGGTGTGTTA ACAAACAATCCTAATTTTGACTACCAATTATTTAATTTGAAC AACTATCGTGCCTTATCAAATAGCACACCCCAAAATAGTITT TCGGAAAAAGTGGATTTAGATAGTTATAGTAGAGGAATGGGC GGACTAGGATTACCTGGAGACTTGTCCTCAATGICTAGATTT GTCAGAGCCGCTTTTACTAAATTAAACTCGTTGTCGATGCAG ACAGAGAGTGGCAGTGTTAGTCAGTTTTTCCATATACTAGGG TCTGTAGAACAACAAAAAGGGCTATGTGAAGTTACTGACGGA AAGTACGAATATACAATCTATTCTTCTTGTTGTGATATGGAC AAAGGAGTTTATTACTATAGAACTTATGACAATAGTCAAATT AACAGTGTCAGTTTAAACCATGAGCACTTGGATACGACTGAA TTAATTTCTTATCCATTACGATCAGAAGCACAATACTATGCA GTTAACTAA Bile salt hydrolase MCTAITYQSYNNYFGRNFDYEISYNEMVTITPRKYPLVFRKV protein from ENLDHHYAIIGITADVESYPLYYDAMNEKGLCIAGLNFAGYA Lactobacillus DYKKYDADKVNITPFELIPWLLGQFSSVREVKKNIQKLNLVN plantarum INFSEQLPLSPLHWLVADKQESIVIESVKEGLKIYDNPVGVL SEQ ID NO: 91 TNNPNFDYQLFNLNNYRALSNSTPQNSFSEKVDLDSYSRGMG GLGLPGDLSSMSRFVRAAFTKLNSLSMQTESGSVSQFFHILG SVEQQKGLCEVTDGKYEYTIYSSCCDMDKGVYYYRTYDNSQI NSVSLNHEHLDTTELISYPLRSEAQYYAVN Bile salt hydrolase ATGTGTACTGCTGCAAATTATTTAACAAAATGCCATTATTTT from GGCCGTAATTTTGACTATGAAATTTCATATAATGAAAGAGTA Methanobrevibacter ACGATAACTCCTAGAAACTATCCTTTAATATTCAGGGATACT smithii 3142 GAGGACATTGAAAATCATTATGGGATTATTGGCATAGCTGCA SEQ ID NO: 92 GGTATTGATGAATATCCTTTGTATTATGATGCATGTAATGAG AAAGGATTAGCTATGGGGGGATTAAACTTTCCGGATTACTGT GACTACAAACCACTAGATAAATCTAAAGTTAACATAGCTTCT TITGAGATTATTCCATATATATTATCTCAAGCAAAAACCATC AGTGATGCCGAAAGGTTATTGGAAAACTTAAATATTTCAGAT GAGAAATTTTCCGCCCAGTTGCCTCCATCTCCACTTCATTGG ATTATTTCAGATAGGAATGCTTCAATTGTTGTAGAGGTTGTA GAGGAAGGACTGGATATTTATGATAATCCTGTAGGAGTTITA ACAAACAACCCTCCTTTTGATAAACAGCTATTTAATTTAAAT AATTATATGGCATTATCAAACAGAACGCCTGAAAATACCTTT GGAGGCAATTTGGATTTGGCAACTTATAGTCGGGGAATGGGT TCAATTGGTCTTCCGGGGGATGTTTCTTCACAGTCCCGTTTT GTAAAAGCAGCTTTTGTTAAAGAAAATTCCGTTTCCGGAGAT TCTGAAAAAGAAAGTGTGTCTCAGTTTTTCCATATTCTGGCA TCTGTTGAACAGCAAAAAGGATGTACGTTAGTGGAAGAACCT GATAAATTTGAGTATACTATTTATTCAGACTGTTACAATACA GATAAGGGAATATTGTATTATAAAACATATGATGGTCCTCAA ACATCTGTTAATATACATGATGAGGATTTGGAAACCAATCAG TTAATTAATTTTGAGTTGGTTGATTAA Bile salt hydrolase MCTAANYLTKCHYFGRNFDYEISYNERVTITPRNYPLIFRDT protein from EDIENHYGIIGIAAGIDEYPLYYDACNEKGLAMGGLNFPDYC Methanobrevibacter DYKPLDKSKVNIASFEIIPYILSQAKTISDAERLLENLNISD smithii 3142 EKFSAQLPPSPLHWIISDRNASIVVEVVEEGLDIYDNPVGVL SEQ ID NO: 93 TNNPPFDKQLFNLNNYMALSNRTPENTFGGNLDLATYSRGMG SIGLPGDVSSQSRFVKAAFVKENSVSGDSEKESVSQFFHILA SVEQQKGCTLVEEPDKFEYTIYSDCYNTDKGILYYKTYDGPQ TSVNIHDEDLETNQLINFELVD Bile salt hydrolase ATGGTTATGAAAAAGATTTTGATAGCTTTGGCCTTATTGCTG from Bacteroides ACAGGCATTGCAAGCGGATCGGCATGTACCGGTATTTCATTC vulgatus CTCGCTGAAGATGGCGGATATGTGCAGGCACGTACTATAGAG SEQ ID NO: 94 TGGGGGAACAGTTATCTTCCGAGTGAATATGTTATTGTTCCC AGAGGACAGGATTTGGTATCTTATACTCCAACGGGTGTAAAT GGCTTGAGATTTCGGGCTAAATATGGTCTGGTAGGACTGGCT ATCATTCAGAAAGAGTTTGTGGCTGAAGGACTGAATGAAGTA GGGCTTTCGGCTGGATTGTTTTATTTTCCCCATTATGGGAAG TATGAAGAATATGATGAGGCTCAAAATGCAATTACTTTGTCG GATTTGCAGGTGGTGAACTGGATGCTTTCCCAATTTGCTACT ATAGACGAAGTGAGAGAAGCTATAGAAGGGGTGAAGGTGGTG TCTCTTGATAAACCTGGTAAAAGTTCTACGGTACATTGGCGC ATTGGCGATGCTAAAGGAAATCAAATGGTGTTGGAATTTGTA GGTGGTGTTCCTTATTTTTATGAAAATAAAGTAGGAGTACTC ACCAATTCTCCCGATTTTCCATGGCAGGTGATTAACTTGAAT AATTATGTAAATCTATATCCGGGAGCTGTCACTCCACAGCAA TGGGGTGGGGTGACTATTTTCCCTTTTGGCGCAGGTGCCGGA TTTCATGGTATTCCGGGGGATGTAACTCCTCCATCCCGTTTT GTTCGTGTAGCGTTTTATAAGGCAACAGCTCCGGTGTGTCCT ACAGCGTATGACGCTATATTACAAAGCTTTCATATCCTGAAT AATTTTGATATTCCTATTGGTATAGAATATGCGTTAGGGAAA GCACCTGATATTCCTAGTGCCACACAATGGACTTCGGCTATT GAITTGACAAACAGGAAAGTGTATTATAAAACAGCATACAAT AACAATATTCGTTGTATTAGTATGAAGAAGATTGATTTTGAT AAAGTGAAGTATCAGTCGTATCCATTGGATAAGGAGTTGAAA CAGCCTGTAGAAGAGATTATTGTGAAATAG Bile salt hydrolase MVMKKILIALALLLTGIASGSACTGISFLAEDGGYVQARTIE protein from WGNSYLPSEYVIVPRGQDLVSYTPTGVNGLRFRAKYGLVGLA Bacteroides IIQKEFVAEGLNEVGLSAGLFYFPHYGKYEEYDEAQNAITLS vulgatus DLQVVNWMLSQFATIDEVREAIEGVKVVSLDKPGKSSTVHWR SEQ ID NO: 95 IGDAKGNQMVLEFVGGVPYFYENKVGVLTNSPDFPWQVINLN NYVNLYPGAVTPQQWGGVTIFPFGAGAGFHGIPGDVTPPSRF VRVAFYKATAPVCPTAYDAILQSFHILNNFDIPIGIEYALGK APDIPSATQWTSAIDLTNRKVYYKTAYNNNIRCISMKKIDFD KVKYQSYPLDKELKQPVEEIIVK Bile salt hydrolase ATGTGCACTGGTGTCCGTTTCTCCGATGATGAGGGCAACAC from CTATTTCGGCCGTAATCTCGACTGGAGTTTCTCATATGGGG Bifidobacterium AGACCATCCTGGTTACTCCGCGCGGCTACCACTATGACACG longum GTGTTTGGTGCGGGCGGCAAGGCGAAGCCGAACGCGGTGAT SEQ ID NO: 96 CGGCGTGGGTGTGGTCATGGCCGATAGGCCGATGTATTTCG ACTGCGCCAATGAACATGGTCTGGCCATCGCCGGCTTGAAT TTCCCCGGCTACGCCTCGTTCGTCCACGAACCGGTCGAAGG CACGGAAAACGTCGCCACGTTCGAATTTCCGCTGTGGGTGG CGCGTAATTTCGACTCCGTCGACGAGGTCGAGGAGGCGCTC AGGAACGTGACGCTCGTCTCCCAGATCGTGCCGGGACAGCA GGAGTCTCTGCTGCACTGGTTCATCGGCGACGGCAAGCGCA GCATCGTCGTCGAGCAGATGGCCGATGGCATGCACGTGCAT CATGATGACGTCGATGTGCTGACCAATCAGCCGACGTTCGA CTTCCATATGGAAAACCTGCGCAACTACATGTGCGTCAGCA ACGAGATGGCCGAACCGACTTCATGGGGCAAGGCCTCCTTG ACCGCCTGGGGTGCGGGTGTGGGCATGCATGGCATCCCGGG CGACGTGAGTTCCCCGTCGCGCTTCGTTCGTGTGGCCTACA CCAACGCGCATTACCCGCAGCAGAACGATGAAGCCGCCAAT GTGTCGCGCCTGTTCCACACCCTCGGCTCCGTGCAGATGGT GGACGGCATGGCGAAGATGGGCGACGGCCAGTTCGAACGCA CGCTGTTCACCAGCGGATATTCGTCCAAGACCAACACCTAT TACATGAACACCTATGATGACCCCGCCATCCGTTCCTACGC CATGGCCGATTACGATATGGATTCCTCGGAGCTCATCAGCG TCGCCCGATGA Bile salt hydrolase MCTGVRFSDDEGNTYFGRNLDWSFSYGETILVTPRGYHYDTV protein from FGAGGKAKPNAVIGVGVVMADRPMYFDCANEHGLAIAGLNFP Bifidobacterium GYASFVHEPVEGTENVATFEFPLWVARNFDSVDEVEEALRNV longum TLVSQIVPGQQESLLHWFIGDGKRSIVVEQMADGMHVHHDDV SEQ ID NO: 97 DVLTNQPTFDFHMENLRNYMCVSNEMAEPTSWGKASLTAWGA GVGMHGIPGDVSSPSRFVRVAYTNAHYPQQNDEAANVSRLFH TLGSVQMVDGMAKMGDGQFERTLFTSGYSSKTNTYYMNTYDD PAIRSYAMADYDMDSSELISVAR Bile salt hydrolase ATGTGTACGTCAATAACTTATACAACGAAGGATCACTATTT from Listeria TGGAAGGAATTTCGATTATGAACTTTCTTACAAAGAAGTTG monocytogenes TGGTTGTTACGCCGAAAAATTACCCGTTCCATTTTCGCAAG SEQ ID NO: 98 GTAGAGGATATAGAGAAGCATTATGCACTTATTGGTATTGC TGCTGTGATGGAAAACTACCCGTTGTATTACGATGCTACCA ATGAAAAAGGCCTTAGTATGGCAGGACTCAATTTCTCAGGA AATGCGGATTACAAGGATTTTGCAGAAGGTAAGGACAATGT GACCCCCTTTGAATTTATTCCGTGGATTCTTGGTCAATGCG CTACTGTAAAAGAAGCAAGAAGATTACTTCAGAGAATCAAT CTCGTGAATATTAGTTTTAGTGAAAATTTACCGCTGTCTCC ATTACATTGGTTGATGGCTGATCAAACAGAATCTATTGTAG TGGAATGTGTGAAAGATGGACTTCACATTTATGATAATCCT GTTGGCGTGTTAACAAATAATCCAACATTTGATTACCAACT ATTTAATTTAAACAATTATCGCGTTCTTTCGAGTGAAACCC CAGAAAATAATTTTTCCAAAGAGATTGATTTGGATGCTTAT AGTCGTGGGATGGGCGGAATTGGCTTACCTGGTGATTTATC TTCTATGTCTCGTTTTGTGAAAGCAACTTTTACCAAATTGA ATTCTGTTTCAGGTGATTCTGAATCAGAAAGTATTAGCCAA TTTTTCCATATTTTAGGCTCGGTGGAACAACAAAAAGGTCT TTGTGATGTTGGTGGGGGAAAATACGAGCATACTATTTATT CCTCGTGTTGCAATATCGATAAAGGAATTTATTATTATAGA ACATACGGAAACAGTCAAATTACTGGTGTGGATATGCACCA AGAGGATTTAGAGAGCAAAGAACTAGCTATTTATCCACTCG TCAATGAGCAACGACTAAACATTGTTAACAAATAA Bile salt hydrolase MCTSITYTTKDHYFGRNFDYELSYKEVVVVTPKNYPFHFRKV protein from EDIEKHYALIGIAAVMENYPLYYDATNEKGLSMAGLNFSGNA Listeria DYKDFAEGKDNVTPFEFIPWILGQCATVKEARRLLQRINLVN monocytogenes ISFSENLPLSPLHWLMADQTESIVVECVKDGLHIYDNPVGVL SEQ ID NO: 99 TNNPTFDYQLFNLNNYRVLSSETPENNFSKEIDLDAYSRGMG GIGLPGDLSSMSRFVKATFTKLNSVSGDSESESISQFFHILG SVEQQKGLCDVGGGKYEHTIYSSCCNIDKGIYYYRTYGNSQI TGVDMHQEDLESKELAIYPLVNEQRLNIVNK Bile salt hydrolase ATGTGTACAGGATTAGCCTTAGAAACAAAAGATGGATTACAT from Clostridium TTGTTTGGAAGAAATATGGATATTGAATATTCATTTAATCAA perfringens TCTATTATATTTATTCCTAGGAATTTTAAATGTGTAAACAAA SEQ ID NO: 100 TCAAACAAAAAAGAATTAACAACAAAATATGCTGTTCTTGGA ATGGGAACTATTTTTGATGATTATCCTACCTTTGCAGATGGT ATGAATGAAAAGGGATTAGGGTGTGCTGGCTTAAATTTCCCT GTTTATGTTAGCTATTCTAAAGAAGATATAGAAGGTAAAACT AATATTCCAGTATATAATTTCTTATTATGGGTTTTAGCTAAT TTTAGCTCAGTAGAAGAGGTAAAGGAAGCATTAAAAAATGCT AATATAGTGGATATACCTATTAGCGAAAATATTCCTAATACA ACTCTTCATTGGATGATAAGCGATATAACAGGAAAGTCTATT GTGGTTGAACAAACAAAGGAAAAATTAAATGTATTTGATAAT AATATTGGAGTATTAACTAATTCACCTACTTTTGATTGGCAT GTAGCAAATTTAAATCAATATGTAGGTTTGAGATATAATCAA GTTCCAGAATTTAAGTTAGGAGATCAATCTTTAACTGCTTTA GGTCAAGGAACTGGTTTAGTAGGATTACCAGGGGACTTTACA CCTGCATCTAGATTTATAAGAGTAGCATTTTTAAGAGATGCA ATGATAAAAAATGATAAAGATTCAATAGACTTAATTGAATTT TTCCATATATTAAATAATGTTGCTATGGTAAGAGGATCAACT AGAACTGTAGAAGAAAAAAGTGATCTTACTCAATATACAAGT TGCATGTGTTTAGAAAAAGGAATTTATTATTATAATACCTAT GAAAATAATCAAATTAATGCAATAGACATGAATAAAGAAAAC TTAGATGGAAATGAAATTAAAACATATAAATACAACAAAACT TTAAGTATTAATCATGTAAATTAG Bile salt hydrolase MCTGLALETKDGLHLFGRNMDIEYSFNQSIIFIPRNFKCVNK protein from SNKKELTTKYAVLGMGTIFDDYPTFADGMNEKGLGCAGLNFP Clostridium VYVSYSKEDIEGKTNIPVYNFLLWVLANFSSVEEVKEALKNA perfringens NIVDIPISENIPNTILHWMISDITGKSIVVEQTKEKLNVFDN SEQ ID NO: 101 NIGVLTNSPTFDWHVANLNQYVGLRYNQVPEFKLGDQSLTAL GQGTGLVGLPGDFTPASRYIRVAFLRDAMIKNDKDSIDLIEF FHILNNVAMVRGSTRTVEEKSDLTQYTSCMCLEKGIYYYNTY ENNQINAIDMNKENLDGNEIKTYKYNKTLSINHVN Bile salt hydrolase ATGTGTACGTCTATTACTTATGTAACAAGTGATCATTATTTT from Enterococcus GGAAGGAATTTTGATTATGAAATATCTTACAATGAAGTAGTT faecium ACTGTTACTCCAAGAAATTATAAGTTGAATTTTCGAAAGGTA SEQ ID NO: 102 AATGATTTGGATACTCATTATGCAATGATTGGTATTGCCGCT GGTATAGCTGACTACCCTCTTTATTACGATGCGACAAATGAA AAAGGATTGAGTATGGCTGGGCTAAATTTTTCTGGGTATGCT GATTATAAAGAAATACAAGAAGGGAAAGACAATGTATCTCCT TTTGAATTTATTCCTTGGATTTTAGGACAATGCTCAACAGTA GGAGAAGCTAAAAAATTGTTAAAAAATATCAATTTAGCAAAT ATAAATTATAGTGACGAACTTCCTTTATCCCCTTTACATTGG CTATTAGCTGATAAAGAAAAATCAATTGTCATTGAAAGTATG AAAGATGGACTTCATATATATGATAACCCTGTGGGCGTTCTT ACCAATAATCCTTCATTTGACTATCAATTATTTAATTTAAAC AATTATCGTGTCTTATCGAGTGAAACTCCTAAAAATAATTTT TCAAATCAAATAAGTTTGAATGCCTATAGCCGCGGTATGGGA GGGATAGGCTTGCCTGGAGATTTATCCTCAGTATCTCGTTTT GTTAAAGCGACTTTTACGAAGCTGAATTCTGTATCTGGAGAT TCAGAGTCAGAAAGTATTAGTCAATTTTTCCATATCTTAGGT TCAGTAGAACAACAAAAAGGTTTGTGTGATGTAGGTGATGGA AAATATGAATATACAATTTATTCTTCTTGTTGCAATGTTGAC AAAGGAATCTATTATTATCGAACATATGAAGACAGTCAAATT ACTGCAATTGATATGAATAAAGAAGACTTAGATAGTCATAAG TTAATTAGTTATCCAATTATAGAAAAACAACAAATTAAATAT ATAAATTAG Bile salt hydrolase MCTSITYVTSDHYFGRNFDYEISYNEVVTVTPRNYKLNFRKV protein from NDLDTHYAMIGIAAGIADYPLYYDATNEKGLSMAGLNFSGYA Enterococcus DYKEIQEGKDNVSPFEFIPWILGQCSTVGEAKKLLKMINLAN faecium INYSDELPLSPLHWLLADKEKSIVIE8MKDGLHIYDNPVGVL SEQ ID NO: 103 TNWPSFDYQLFNLNNYRVLSSETPKNNFSNQISLNAYSRGMG GIGLPGDLSSVSRFVKATFTKLNSVSGDSESESISQFFHILG SVEQQKGLCDVGDGKYEYTIYSSCCNVDKGIYYYRTYEDSQI TAIDMNKEDLDSHKLISYPIIEKQQIKYIN Bile salt hydrolase AAGAGAAAAATATGTGTACATCAATTATATTCAGTCCCAAAG A from ATCATTACTTTGGTCGTAACCTTGATTTAGAAATTACTTTTG Lacotbacillus GTCAACAAGTTGTTATTACGCCACGCAATTACACTTTTAAAT acidophilus TCCGTAAGATGCCCAGTTTAAAAAAGCACTATGCAATGATTG SEQ ID NO: 104 GTATCTCATTAGATATGGATGATTATCCCCTATATTTCGACG CTACAAATGAAAAAGGTTTAGGTATGGCCGGACTCAACTATC CAGGAAATGCTACATATTATGAAGAAAAAGAAAATAAAGATA ATATTGCTTCCTTTGAATTCATCCCTTGGATTTTAGGACAGT GTAGCACTATTAGCGAAGTAAAGGATTTACTTAGCAGAATCA ACATCGCCGATTTAAATTTCAGCGAAAAAATGCAAGCCTCCT CTCTTCACTGGCTTATTGCAGATAAAACAGGTACATCATTAG TTGTTGAAACAGACAAAGATGGAATGCATATTTATGATAATC CAGTTGGCTGCTTAACTAATAATCCACAATTTCCAAAGCAAT TATTCAATTTAAATAACTATGCTGACGTATCTCCAAAAATGC CTAAAAATAACTTCTCAGATAAAGTAAATATGGCTGGCTACA GCCGTGGATTAGGGTCTCACAACTTACCAGGTGGAATGGATT CTGAATCACGTTTTGTCAGAGTAGCTTTCAATAAATTTAATG CTCCAATTGCTGAAACCGAAGAAGAAAATATTGATACTTACT TCCACATTTTACATTCGGTTGAACAACAAAAGGGACTGGATG AAGTTGGTCCAAACTCATTTGAATATACAATTTATTCTGATG GAACTAACTTAGACAAAGGTATTTTCTACTACACCACTTATT CAAACAAACAAATTAACGTTGTTGATATGAATAAAGAAGATC TAGATAGCAGCAATTTGATCACTTATGATATGCTTGATAAAA CTAAATTTAACCATCAAAACTAA Bile salt hydrolase MCTSIIFSPKDHYFGRNLDLEITFGQQVVITPRNYTFKFRKM A protein from PSLKKHYAMIGISLDMDDYPLYYDATNEKGLGMAGINYPGNA Lacotbacillus TYYEEKENKDNIASFEFIPWILGQCSTISEVKDLLSRINIAD acidophilus LNFSEKMQASSLHWLIADKIGTSLVVETDKDGMHIYDNPVGC SEQ ID NO: 105 LTNNPQFPKQLFNLNNYADVSPKMPKNNFSDKVNMAGYSRGL GSHNLPGGMDSESRFVRVAFNKFNAPIAETEEENIDTYFHIL HSVFOQKGLDEVGPNSFEYTIYSDGINLDKGIFYYTTYSNKQ INVVDMNKFDLDSSNIITYDPILDKIKFNHQN Bilesalthydrolase AGAAAGCGTGCAGTAAATGTGTACATCAATTTGTTATAATC B from CTAACGATCATTATTTTGGTAGAAATCTTGACTATGAAATT Lacotbacillus GCTTATGGTCAAAAAGTAGTCATTGTACCAAGAAACTACGA acidophilus ATTTAAGTATAGAGAAATGCCCTCTCAAAAGATGCATTATG SEQ ID NO: 106 CTTTTATCGGAGTATCTGTAGTTAATGATGATTATCCATTA TTATGTGATGCAATTAATGAAAAGGGGCTTGGTATTGCAGG ATTAAATTTTCAAGGTCCTAATCATTACTTTCCTAAAATCG AAGGTAAGAAGAATATTGCTTCTTTTGAATTAATGCCATAC TTATTAAGTAATTGTGAAAATACTGACGATGTTAAAGAAAT CTTAGATAATGCAAATATTTTAAATATTAGCTTTTCAGCAA ATTATCCTGCAGCTGATTTACATTGGATTTTAAGTGATAAA GCTGGTAAGAGTATCGTAGTTGAATCAACCAATTCAGGTTT ACATATTTATGATAATCCAGTGAATGTCTTAACTAACAATC CTGAATTTCCGGATCAATTAATTAAATTAAGTGACTACGCC GACGTTACTCCACATAATCCTAAGAATACATTGGTTCCTAA TGTTGATCTTAATCTATATAGTAGAGGCTTAGGTACTCACC ACTTACCTGGTGGAATGGATTCTAGCTCTCGATTTGTTAAG GTAGCTTTTGTCTTGGCACACACTCCACAAGGAAAAAATGA AGTGGAAAATGTTACTAATTATTTCCATATTCTGCATTCAG TAGAACAACCTGATGGTTTAGATGAAGTAGAAGATAATCGC TATGAATATACTATGTATACAGATTGTATGAACTTAGATAA AGGTATTTTGTACTTTACTACTTATGACAATAATCGGATTA ATGCAGTAGATATGCATAAAGCAGATTTAGATTCAGAAGAT TTAATCTGCTACGATTTGTTTAAGAAACAAGATATTGAATA TATGAATTAA Bile salt hydrolase MCTSICYNPNDHYFGRNLDYEIAYGQKVVIVPRNYEFKYREM B protein from PSQKMHYAFIGVSVVNDDYPLLCDAINEKGLGIAGLNFQGPN Lacotbacillus HYFPKIEGKKNIASFELMPYLLSNCENTDDVKEILDNANILM acidophilus ISFSANYPAADLHWILSDKAGKSIVVESTNSGLHIYDNPVNV SEQ ID NO: 107 LTNNPEFPDQLIKLSDYADVTPHNPKNTLVPNVDLNLYSRGL GTHHLPGGMDSSSRFVKVAFVLAHTPQGKNEVENVTNYFHIL HSVEQPDGLDEVEDNRYEYTMYTDCMNLDKGILYFTTYDNNR INAVDMHKADLDSEDLICYDLFKKQDIEYMN Bile salt hydrolase ATGGAAACGAAAAGCTCTCTCTGGAAATCATCGCGCCGCGT from Brucella GCTTGCACATGGGGCTGCAACTGTTCTGGTCGCGGCGGGCC abortus TTATCGTTCCCCAGGCGGCTATGGCTTGCACGAGCTTCGTT SEQ ID NO: 108 CTGCCGACGAGCGACGGTGGTATGGTCTATGGTCGCACGAT GGAATTCGGGTTCAATCTCAAATCCGACATGATTGCCATTC CGCGCAATTACACCATCACGGCAAGCGGGCCGGACGGTGCT GCGGGCAAGAAATGGAAGGGCAAATATGCCACGATCGGCAT GAATGCTTTTGGTATCGTCGCTCTCACCGACGGTATGAACG AGAAGGGGCTTGCAGGCGGGCTTCTCTATTTCCCGGAATAT GCCAAGTATCAGGACCCATCCACGGCGAAGCCGGAAGACAG CCTCGCTCCGTGGGATTTCCTGACCTGGGCGCTGGCCAATT TTTCGACAGTGGCCGAAGTCAAGGATGCTTTGAGCACCATT TCCATCGTCGATGTGAAACAAAAGGACCTGGGATTTACCCC GCCCGCTCACTACACGCTGCATGATGCGACCGGCGCATCCA TCGTGATCGAACCGATCGACGGCAAGCTCAAGGTTTACGAC AACAAGCTCGGTGTCATGACCAATTCGCCGTCTTTCGACTG GCACATGACCAATCTGCGCAACTATGTCTATCTCTCGCGTG AAAATCCGAAGCCGTTGCAGATCCTTGGCGAGACGATCCAG TCATTCGGGCAAGGCGCCGGTATGCATGGTATTCCGGGCGA CACCACGCCGCCATCGCGTTTCGTGCGTGCAAGCGCCTACG TCCTTTCCGCCAAGAAGGTGCCGAGCGGCCTTGAAAGCGTG CGGCTGGCCGAGCATATTGCCAATAACTTCGACATTCCAAA GGGATGGAGCGAAGAGCAGAATATGTTTGAATATACCCAGT GGACCGCCTTTGCGGACATGAAGAACGATGTCTATTACATC AAGACCTATGACGATCAGGTTCTGCGCAGCTTCAGCTTCAA GGATTTTGATGTCGATAGCAAAGATATTCTAACGATCAAGT TCGAGCCAAAACTGGACGCGCCGTCACTGAAAAAGTAA Bile salt hydrolase METKSSLWKSSRRVLAHGAATVLVAAGLIVPQAAMACTSFVL protein from PTSDGGMVYGRTMEFGFNLKSDMIAIPRNYTITASGPDGAAG Brucella abortus  KKWKGKYATIGMNAFGIVALTDGMNEKGLAGGLLYFPEYAKY SEQ ID NO: 109 QDPSTAKPEDSLAPWDFLTWALANFSTVAEVKDALSTISIVD VKQKDLGFTPPAHYTLHDATGASIVIEPIDGKLKVYDNKLGV MTNSPSFDWHMTNLRNYVYLSRENPKPLQILGETIQSFGQGA GMHGIPGDTTPPSRFVRASAYVLSAKKVPSGLESVRLAEHIA NNFDIPKGWSEEQNMFEYTQWTAFADMKNDVYYIKTYDDQVL RSFSFKDFDVDSKDILTIKFEPKLDAPSLKK

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 90. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 90. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 90. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 90. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 90.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 92. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 92. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 92. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 92. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 92.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 94 In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 94. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 93. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 94. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 94. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 94. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 94. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 94.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 96 In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 96. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 96. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 96. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 96. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 96. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 96. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 96.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 98. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 98. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 98. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 98. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 98.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 100. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 100. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 100. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 100. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 100.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 102. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 102. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 102. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 102. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 102.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 104. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 104. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 104. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 104. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 104.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 106. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 106. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 106. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 106. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 106.

In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 108. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 108. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 108. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 108. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 108.

In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109.

In one embodiment, the gene encoding the bile salt hydrolase enzyme is directly operably linked to a first promoter. In another embodiment, the gene encoding the bile salt hydrolase enzyme is indirectly operably linked to a first promoter. In one embodiment, the gene encoding bile salt hydrolase enzyme is operably linked to a promoter that it is not nauturally linked to in nature.

In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a constitutive promoter. In another embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the bile salt hydrolase enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.

In some embodiments, the genetically engineered bacteria are capable of expressing bile sale hydrolase under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of expressing bile sale hydrolase in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose .

The gene encoding the bile salt hydrolase enzyme may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the bile salt hydrolase is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding a bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located in the chromosome of the bacterial cell. For example, E. coli comprises a native bile salt hydrolase gene.

In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a low-copy plasmid. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the bile salt hydrolase enzyme, thereby increasing the catabolism of bile salts.

Transporters of Bile Salts and Bile Acids

The uptake of bile salts into the Lactobacillus and Bifidobacterium has been found to occur via the bile salt transporters CbsT1 and CbsT2 (see, e.g., Elkins et al., Microbiology, 147(Pt. 12):3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference). The uptake of bile acids into the Neisseria meningitides has been found to occur via the bile acid sodium symporter ASBT (see, e.g., Hu et al., Nature, 478(7369):408-411 (2011), the contents of which are expressly incorporated herein by reference. Other proteins that mediate the import of bile salts or acids into cells are well known to those of skill in the art. For the purposes of this invention, a bile salt transporter includes bile salt importers and bile acid symporters.

Bile salt transporters, e.g., bile salt importers or bile acid symporters, may be expressed or modified in the bacteria in order to enhance bile salt or acid transport into the cell. Specifically, when the transporter of bile salts is expressed in the recombinant bacterial cells, the bacterial cells import more bile salts into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a transporter of bile salts may be used to import bile salts into the bacteria so that any gene encoding a bile salt hydrolase (BSH) enzyme expressed in the organism can be used to treat disorders associated with bile salts, such as cardiac disease, metabolic disease, liver disease, cancer, and C. difficile infection. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt and a heterologous gene encoding a bile salt hydrolase (BSH) enzyme.

Thus, in some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a transporter of a bile salt. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a transporter of a bile salt operably linked to the first promoter. In another embodiment, the disclosure provides a bacterial cell that comprises a heterologous gene encoding at least one bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding transporter of a bile salt operably linked to a second promoter. In one embodiment, the first promoter and the second promoter are separate copies of the same promoter. In another embodiment, the first promoter and the second promoter are different promoters. In some embodiments the gene encoding at least one bile salt hydrolase enzyme and/or the heterologous gene encoding transporter of a bile salt are operably linked to a promoter that it is not naturally linked to in nature.

In one embodiment, the bacterial cell comprises a gene encoding a transporter of a bile salt from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one native gene encoding transporter of a bile salt. In some embodiments, the at least one native gene encoding atransporter of a bile salt is not modified. In another embodiment, the bacterial cell comprises more than one copy of at least one native gene encoding a transporter of a bile salt. In yet another embodiment, the bacterial cell comprises a copy of a gene encoding a native transporter of a bile salt, as well as at least one copy of a heterologous gene encoding a transporter of a bile salt from a different bacterial species. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of the heterologous gene encoding a tarnsporter of a bile salt. In one embodiment, the bacterial cell comprises multiple copies of the heterologous gene encoding a transporter of a bile salt.

In some embodiments, the transporterof a bile salt is encoded by a transporter of a bile salt gene derived from a bacterial genus or species, including but not limited to, Lactobacillus. In some embodiments, the transporterof a bile salt gene is derived from a bacteria of the species Lactobacillus johnsonni strain 100-100.

The present disclosure further comprises genes encoding functional fragments of a transporter of a bile salt or functional variants of a transporter of a bile salt. As used herein, the term “functional fragment thereof” or “functional variant thereof” of a transporter of a bile salt relates to an element having qualitative biological activity in common with the wild-type transporter of a bile salt from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated transporter of bile salt protein is one which retains essentially the same ability to import the bile salt into the bacterial cell as does the transporter protein from which the functional fragment or functional variant was derived. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional fragment of a transporter of a bile salt. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional variant of a transporter of a bile salt.

Assays for testing the activity of a transporter of a bile salt, a functional variant of a transporter of a bile salt, or a functional fragment of a transporter of a bile salt are well known to one of ordinary skill in the art. For example, bile salt import can be assessed as described in Elkins et al., Microbiology, 147:3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference.

In one embodiment, the gene(s) encoding the transporter of a bile salt have been codon-optimized for use in the host organism. In one embodiment, the genes encoding the transporter of a bile salt have been codon-optimized for use in Escherichia coli.

The present disclosure also encompasses genes encoding a transporter of a bile salt comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.

In some embodiments, the gene encoding a transporter of a bile salt is mutagenized; mutants exhibiting increased bile salt transport are selected; and the mutagenized a gene encoding a transporter of a bile salt is isolated and inserted into the bacterial cell. In some embodiments, the gene encoding a transporter of a bile salt is mutagenized; mutants exhibiting decreased bile salt transport are selected; and the mutagenized a gene encoding a transporter of the bile salt is isolated and inserted into the bacterial cell. The transporter modifications described herein may be present on a plasmid or chromosome. Non-limiting examples of bile salt transporters, which are encoded in the genetically engineered bacteria, are in Table 11B.

TABLE 11B Bile Salt Transport and Export Sequences Description Sequence cbsT1 from ATGTCGACCACACCGACACAGCCATCATCACGAAAACAG Lactobacillus GCTGTTTACCCGTACTTGATCGTGCTGTCGGGCATCGTCT johnsonii TCACGGCCATCCCGGTATCGCTGGTCTGCAGTTGCGCAGG SEQ ID NO: TATCTTCTTCACGCCTGTCAGCAGCTACTTCCATGTTCCCA 110 AGGCCGCATTCACCGGATATTTCAGCATATTCAGCATCAC CATGGTCGCCTTCCTGCCGGTGGCCGGATGGCTGATGCAC CGCTACGATCTGCGCATCGTACTGACCGCAAGCACCGTCC TGGCTGGACTGGGCTGCCTGGGTATGTCCCGATCATCCGC CATGTGGCAGTTCTATCTATGCGGAGTGGTTCTGGGAATC GGCATGCCGGCCGTCCTCTATCTGTCAGTGCCAACACTCA TCAACGCCTGGTTCCGCAAGCGGGTCGGGTTCTTCATCGG CCTGTGCATGGCCTTCACCGGCATAGGCGGCGTGATCTTC AACCAGATAGGCACCATGATCATCAGATCCGCCCCTGAT GGATGGAGGCGGGGATATCTGGTTTTCGCTATTCTCATCC TGGTGATCACCCTGCCCTTCACCATTTTCGTCATTCGCAG CACACCCGAACAGATGGGTCTGCATCCCTACGGCGCCGA CCAGGAGCCTGATGCAGCTGAGACGGCCACCAATAGTGC AGGCACCGGGAGCAAAGACCAAAAGAGTCCTGAGCCTGC AGCGTCAACCGTAGGCATGACTGCCTCCCAGGCCTTGCGC TCCCCTGCCTTCTGGGCGCTGGCGCTCTTCTGCGGTCTGA TCACCATGAATCAGACCATTTACCAGTTCCTGCCCTCCTA CGCGGCATCCCTGCCATCCATGGCAGCCTACACGGGACT GATCGCCTCCTCCTGCATGGCCGGCCAGGCCATCGGCAA GATCATCCTGGGCATGGTCAACGACGGCAGCATCGTAGG CGGTCTCTGTCTGGGCATCGGCGGCGGCATTCTCGGCGTC TGCCTCATGGTCGCCTTCCCCGGATTGCCCGTGCTCCTCCT GCTGGGAGCCTTTGCCTTCGGCCTTGTCTACGCCTGCACT ACTGTGCAGACACCAATCCTGGTTACAGCGGTCTTCGGCT CGCGCGACTACACCAACATCTATGCACGTATCCAGATGGT TGGGTCCCTAGCCTCGGCCTTCGCAGCTCTCTTCTGGGGC GCCATCGCTGACCAGCCCCACGGCTACATCATCATGTTCG GTCTGAGCATCCTGATCATGGTTGTGGCCTTGTTCCTAGG CATTATCCCTCTGAAAGGTACGCGCAAGTTGACCGATCAG ATCGCCTGA CbsT1 protein MSTTPTQPSSRKQAVYPYLIVLSGIVFTAIPVSLVCSCAGIFFT Lactobacillus PVSSYFHVPKAAFTGYFSIFSITMVAFLPVAGWLMHRYDLRI johnsonii VLTASTVLAGLGCLGMSRSSAMWQFYLCGVVLGIGMPAVL SEQ ID NO: YLSVPTLINAWFRKRVGFFIGLCMAFTGIGGVIFNQIGTMIIR 111 SAPDGWRRGYLVFAILILVTTLPFTIFVIRSTPEQMGLHPYGA DQEPDAAETATNSAGTGSKDQKSPEPAASTVGMTASQALRS PAFWALALFCGLITMNQTIYQFLPSYAASLPSMAAYTGLIAS SCMAGQAIGKIILGMVNDGSIVGGLCLGIGGGILGVCLMVAF PGLPVLLLLGAFAFGLVYACTTVQTPILVTAVFGSRDYTNIY ARIQMVGSLASAFAALFWGAIADQPHGYIIMFGLSILIMVVA LFLGHPLKGTRKLTDQIA cbsT2 from ATGTCTACTGATGCCGCTACTAAAGATAAAGTAGTAAGC Lactobacillus AAGGGCTATAAATACTTCATGGTTTTCCTTTGTATGTTAA Johnsonii CCCAAGCTATTCCTTATGGAATTGCTCAAAACATTCAGCC SEQ ID NO: TTTGTTTATCCACCCTTTAGTTAATACTTTCCACTTTACCT 112 TAGCATCGTACACATTAATTTTTACGTTTGGTGCGGTTTTT GCTTCAGTTGCTTCTCCATTTATTGGTAAGGCATTAGAAA AAGTTAACTTCCGACTAATGTATTTAATTGGTATTGGTCT TTCTGCTATTGCCTACGTAATTTTTGGAATTAGTACAAAA CTACCCGGTTTCTATATTGCCGCTATCATTTGTATGGTTGG TTCAACCTTTTACTCCGGCCAAGGTGTTCCCTGGGTTATT AACCACTGGTTCCCAGCAAAGGGACGTGGGGCTGCCTTA GGAATTGCCTTCTGCGGTGGTTCTATTGGTAATATCTTTTT ACAACCAGCAACCCAAGCTATTTTAAAACACTACATGAC AGGTAATACTAAGACCGGTCATTTAACCTCTATGGCACCA TTCTTTATCTTTGCCGTAGCTTTATTAGTAATCGGTGTAAT TATCGCCTGCTTCATTAGAACCCCTAAGAAAGACGAAATT GTTGTTTCTGATGCAGAACTAGCTGAAAGCAAGAAAGCT GAAGCCGCAGCCAAAGCTAAAGAGTTTAAAGGCTGGACT AGTAAACAAGTGTTACAAATGAAATGGTTCTGGATTTTCA GCCTTGGTTTCTTAATCATTGGTTTAGGCTTAGCTTCTTTA AATGAAGACTATGCCGCCTTCCTTGATACTAAGCTTTCTT TAACCGATGTTGGTTTAGTTGGGTCAATGTACGGTGTTGG TTGTTTAATCGGAAATATTTCTGGTGGTTTCTTATTTGATA AATTTGGTACAGCAAAATCAATGACCTATGCTGGTTGTAT GTATATTTTATCTATTCTGATGATGATCTTTATTAGCTTCC AGCCATATGGTTCATCTATTAGTAAGGCTGCTGGCATTGG CTATGCTATCTTTTGCGGCTTAGCTGTATTTAGTTACATGT CAGGCCCAGCCTTCATGGCAAAAGACCTCTTTGGTTCAAG AGATCAAGGTGTCATGCTTGGATACGTTGGTTTAGCTTAT GCAATTGGCTATGCCATTGGTGCTCCACTATTTGGGATTA TTAAGGGAGCGGCAAGCTTTACAGTTGCTTGGTACTTTAT GATTGCCTTTGTTGCAATTGGTTTTATCATTTTAGTATTTG CCGTTATCCAAATTAAGAGATACCAAAAGAAATACATTG CAGAGCAAGCAGCAAAAGCTAATGCTAAATAA CbsT2 protein MSTDAATKDKVVSKGYKYFMVFLCMLTQAIPYGIAQNIQPL from FIHPLVNTFHFTLASYTLIFTFGAVFASVASPFIGKALEKVNF Lactobacillus RLMYLIGIGLSAIAYVIFGISTKLPGFYIAAIICMVGSTFYSGQ Johnsonii GVPWVINHWFPAKGRGAALGIAFCGGSIGNIFLQPATQAILK SEQ ID NO: HYMTGNTKTGHLTSMAPFFIFAVALLVIGVIIACFIRTPKKDE 113 IVVSDAELAESKKABAAAKAKEFKGWTSKQVLQMKWFWIF SLGFLIIGLGLASLNEDYAAFLDTKLSLTDVGLVGSMYGVG CLIGNISGGFLFDKFGTAKSMTYAGCMYILSILMMIFISFQPY GSSISKAAGIGYAIFCCLAVFSYMSGPAFMAKDLFGSRDQG VMLGYVGLAYAIGYAIGAPLFGIIKGAASFTVAWYFMIAFV AIGFIILVFAVIQIKRYQKKYIABQAAKANAK ABCB11 bile GAATGATGAAAACCGAGGTTGGAAAAGGTTGTGAAACCT salt exporter TTTAACTCTCCACAGTGGAGTCCATTATTTCCTCTGGCTTC Homo sapiens CTCAAATTCATATTCACAGGGTCGTTGGCTGTGGGTTGCA SEQ ID NO: ATTACCATGTCTGACTCAGTAATTCTTCGAAGTATAAAGA 114 AATTTGGAGAGGAGAATGATGGTTTTGAGTCAGATAAAT CATATAATAATGATAAGAAATCAAGGTTACAAGATGAGA AGAAAGGTGATGGCGTTAGAGTTGGCTTCTTTCAATTGTT TCGGTTTTCTTCATCAACTGACATTTGGCTGATGTTTGTGG GAAGTTTGTGTGCATTTCTCCATGGAATAGCCCAGCCAGG CGTGCTACTCATTTTTGGCACAATGACAGATGTTTTTATT GACTACGACGTTGAGTTACAAGAACTCCAGATTCCAGGA AAAGCATGTGTGAATAACACCATTGTATGGACTAACAGT TCCCTCAACCAGAACATGACAAATGGAACACGTTGTGGG TTGCTGAACATCGAGAGCGAAATGATCAAATTTGCCAGTT ACTATGCTGGAATTGCTGTCGCAGTACTTATCACAGGATA TATTCAAATATGCTTTTGGGTCATTGCCGCAGCTCGTCAG ATACAGAAAATGAGAAAATTTTACTTTAGGAGAATAATG AGAATGGAAATAGGGTGGTTTGACTGCAATTCAGTGGGG GAGCTGAATACAAGATTCTCTGATGATATTAATAAAATCA ATGATGCCATAGCTGACCAAATGGCCCTTTTCATTCAGCG CATGACCTCGACCATCTGTGGTTTCCTGTTGGGATTTTTCA GGGGTTGGAAACTGACCTTGGTTATTATTTCTGTCAGCCC TCTCATTGGGATTGGAGCAGCCACCATTGGTCTGAGTGTG TCCAAGTTTACGGACTATGAGCTGAAGGCCTATGCCAAA GCAGGGGTGGTGGCTGATGAAGTCATTTCATCAATGAGA ACAGTGGCTGCTTTTGGTGGTGAGAAAAGAGAGGTTGAA AGGTATGAGAAAAATCTTGTGTTCGCCCAGCGTTGGGGA ATTAGAAAAGGAATAGTGATGGGATTCTTTACTGGATTCG TGTGGTGTCTCATCTTTTTGTGTTATGCACTGGCCTTCTGG TACGGCTCCACACTTGTCCTGGATGAAGGAGAATATACA CCAGGAACCCTTGTCCAGATTTTCCTCAGTGTCATAGTAG GAGCTTTAAATCTTGGCAATGCCTCTCCTTGTTTGGAAGC CTTTGCAACTGGACGTGCAGCAGCCACCAGCATTTTTGAG ACAATAGACAGGAAACCCATCATTGACTGCATGTCAGAA GATGGTTACAAGTTGGATCGAATCAAGGGTGAAATTGAA TTCCATAATGTGACCTTCCATTATCCTTCCAGACCAGAGG TGAAGATTCTAAATGACCTCAACATGGTCATTAAACCAG GGGAAATGACAGCTCTGGTAGGACCCAGTGGAGCTGGAA AAAGTACAGCACTGCAACTCATTCAGCGATTCTATGACCC CTGTGAAGGAATGGTGACCGTGGATGGCCATGACATTCG CTCTCTTAACATTCAGTGGCTTAGAGATCAGATTGGGATA GTGGAGCAAGAGCCAGTTCTGTTCTCTACCACCATTGCAG AAAATATTCGCTATGGCAGAGAAGATGCAACAATGGAAG ACATAGTCCAAGCTGCCAAGGAGGCCAATGCCTACAACT TCATCATGGACCTGCCACAGCAATTTGACACCCTTGTTGG AGAAGGAGGAGGCCAGATGAGTGGTGGCCAGAAACAAA GGGTAGCTATCGCCAGAGCCCTCATCCGAAATCCCAAGA TTCTGCTTTTGGACATGGCCACCTCAGCTCTGGACAATGA GAGTGAAGCCATGGTGCAAGAAGTGCTGAGTAAGATTCA GCATGGGCACACAATCATTTCAGTTGCTCATCGCTTGTCT ACGGTCAGAGCTGCAGATACCATCATTGGTTTTGAACATG GCACTGCAGTGGAAAGAGGGACCCATGAAGAATTACTGG AAAGGAAAGGTGTTTACTTCACTCTAGTGACTTTGCAAAG CCAGGGAAATCAAGCTCTTAATGAAGAGGACATAAAGGA TGCAACTGAAGATGACATGCTTGCGAGGACCTTTAGCAG AGGGAGCTACCAGGATAGTTTAAGGGCTTCCATCCGGCA ACGCTCCAAGTCTCAGCTTTCTTACCTGGTGCACGAACCT CCATTAGCTGTTGTAGATCATAAGTCTACCTATGAAGAAG ATAGAAAGGACAAGGACATTCCTGTGCAGGAAGAAGTTG AACCTGCCCCAGTTAGGAGGATTCTGAAATTCAGTGCTCC AGAATGGCCCTACATGCTGGTAGGGTCTGTGGGTGCAGC TGTGAACGGGACAGTCACACCCTTGTATGCCTTTTTATTC AGCCAGATTCTTGGGACTTTTTCAATTCCTGATAAAGAGG AACAAAGGTCACAGATCAATGGTGTGTGCCTACTTTTTGT AGCAATGGGCTGTGTATCTCTTTTCACCCAATTTCTACAG GGATATGCCTTTGCTAAATCTGGGGAGCTCCTAACAAAA AGGCTACGTAAATTTGGTTTCAGGGCAATGCTGGGGCAA GATATTGCCTGGTTTGATGACCTCAGAAATAGCCCTGGAG CATTGACAACAAGACTTGCTACAGATGCTTCCCAAGTTCA AGGGGCTGCCGGCTCTCAGATCGGGATGATAGTCAATTC CTTCACTAACGTCACTGTGGCCATGATCATTGCCTTCTCCT TTAGCTGGAAGCTGAGCCTGGTCATCTTGTGCTTCTTCCC CTTCTTGGCTTTATCAGGAGCCACACAGACCAGGATGTTG ACAGGATTTGCCTCTCGAGATAAGCAGGCCCTGGAGATG GTGGGACAGATTACAAATGAAGCCCTCAGTAACATCCGC ACTGTTGCTGGAATTGGAAAGGAGAGGCGGTTCATTGAA GCACTTGAGACTGAGCTGGAGAAGCCCTTCAAGACAGCC ATTCAGAAAGCCAATATTTACGGATTCTGCTTTGCCTTTG CCCAGTGCATCATGTTTATTGCGAATTCTGCTTCCTACAG ATATGGAGGTTACTTAATCTCCAATGAGGGGCTCCATTTC AGCTATGTGTTCAGGGTGATCTCTGCAGTTGTACTGAGTG CAACAGCTCTTGGAAGAGCCTTCTCTTACACCCCAAGTTA TGCAAAAGCTAAAATATCAGCTGCACGCTTTTTTCAACTG CTGGACCGACAACCCCCAATCAGTGTATACAATACTGCA GGTGAAAAATGGGACAACTTCCAGGGGAAGATTGATTTT GTTGATTGTAAATTTACATATCCTTCTCGACCTGACTCGC AAGTTCTGAATGGTCTCTCAGTGTCGATTAGTCCAGGGCA GACACTGGCGTTTGTTGGGAGCAGTGGATGTGGCAAAAG CACTAGCATTCAGCTGTTGGAACGTTTCTATGATCCTGAT CAAGGGAAGGTGATGATAGATGGTCATGACAGCAAAAAA GTAAATGTCCAGTTCCTCCGCTCAAACATTGGAATTGTTT CCCAGGAACCAGTGTTGTTTGCCTGTAGCATAATGGACAA TATCAAGTATGGAGACAACACCAAAGAAATTCCCATGGA AAGAGTCATAGCAGCTGCAAAACAGGCTCAGCTGCATGA TTTTGTCATGTCACTCCCAGAGAAATATGAAACTAACGTT GGGTCCCAGGGGTCTCAACTCTCTAGAGGGGAGAAACAA CGCATTGCTATTGCTCGGGCCATTGTACGAGATCCTAAAA TCTTGCTACTAGATGAAGCCACTTCTGCCTTAGACACAGA AAGTGAAAAGACGGTGCAGGTTGCTCTAGACAAAGCCAG AGAGGGTCGGACCTGCATTGTCATTGCCCATCGCTTGTCC ACCATCCAGAACGCGGATATCATTGCTGTCATGGCACAG GGGGTGGTGATTGAAAAGGGGACCCATGAAGAACTGATG GCCCAAAAAGGAGCCTACTACAAACTAGTCACCACTGGA TCCCCCATCAGTTGACCCAATGCAAGAATCTCAGACACAC ATGACGCACCAGTTACAGGGGTTGTTTTTAAAGAAAAAA ACAATCCCAGCAGGAGGGATTGCTGGGATTGTTTTTTCTT TAAAGAAGAATGTTAATATTTTACTTTTACAGTCATTTTC CTACATCGGAATCCAAGCTAATTTCTAATGGCCTTCCATA ATAATTCTGCTTTAGATGTGTATACAGAAAATGAAAGAA ACTAGGGTCCATATGAGGGAAAACCCAATGTCAAGTGGC AGCTCAGCCACCACTCAGTGCTTCTCTGTGCAGGAGCCAG TCCTGATTAATATGTGGGAATTAGTGAGACATCAGGGAG TAAGTGACACTTTGAACTCCTCAAGGGCAGAGAACTGTCT TTCATTTTTGAACCCTCGGTGTACACAGAGGCGGGTCTAT AACAGGCAATCAACAAACGTTTCTTGAGCTAGACCAAGG TCAGATTTGAAAAGAACAGAAGGACTGAAGACCAGCTGT GTTTCTTAACTAAATTTGTCTTTCAAGTGAAACCAGCTTC CTTCATCTCTAAGGCTAAGGATAGGGAAAGGGTGGATGC TCTCAGGCTGAGGGAGGCAGAAAGGGAAAGTATTAGCAT GAGCTTTCCAGTTAGGGCTGTTGATTTATGCTTTAACTTC AGAGTGAGTGTAGGGGTGGTGATGCT ABCB11 bile MSDSVILRSIKKFGEENDGFESDKSYNNDKKSRLQDEKKGD salt exporter GVRVGFFQLFRFSSSTDIWLMFVGSLCAFLHGIAQPGVLLIF protein Homo GTMTDVFIDYDVELQELQIPGKACVNNTIVWTNSSLNQNMT sapiens NGTRCGLLNIESEMIKFASYYAGIAVAVLITGYIQICFWVIAA SEQ ID NO: ARQIQKMRKFYFRRIMRMEIGWFDCNSVGELNTRFSDDINKI 115 NDAIADQMALFIQRMTSTICGFLLGFFRGWKLTLVIISVSPLI GIGAATIGLSVSKFTDYELKAYAKAGVVADEVISSMRTVAA FGGEKREVERYEKNLVFAQRWGIRKGIVMGFFTGFVWCLIF LCYALAFWYGSTLVLDEGEYTPGTLVQIFLSVIVGALNLGN ASPCLEAFATGRAAATSIFETIDRKPIIDCMSEDGYKLDRIKG EIEFHNVTFHYPSRPEVKILNDLNMVIKPGEMTALVGPSGAG KSTALQLIQRFYDPCEGMVTVDGHDIRSLNIQWLRDQIGIVE QEPVLFSTTIAENIRYGREDATMEDIVQAAKEANAYNFIMDL PQQFDTLVGEGGGQMSGGQKQRVAIARALIRNPKILLLDMA TSALDNESEAMVQEVLSKIQHGHTIISVAHRLSTVRAADTIIG FEHGTAVERGTHEELLERKGVYFTLVTLQSQGNQALNEEDI KDATEDDMLARTFSRGSYQDSLRASIRQRSKSQLSYLVHEPP LAVVDHKSTYEEDRKDKDIPVQEEVEPAPVRRILKFSAPEWP YMLVGSVGAAVNGTVTPLYAFLFSQILGTFSIPDKEEQRSQI NGVCLLFVAMGCVSLFTQFLQGYAFAKSGELLTKRLRKFGF RAMLGQDIAWFDDLRNSPGALTTRLATDASQVQGAAGSQI GMIVNSFTNVTVAMIIAFSFSWKLSLVILCFFPFLALSGATQT RMLTGFASRDKQALEMVGQITNEALSNIRTVAGIGKERRFIE ALETELEKPFKTAIQKANIYGFCFAFAQCIMFIANSASYRYG GYLISNEGLHFSYVFRVISAVVLSATALGRAFSYTPSYAKAK ISAARFFQLLDRQPPISVYNTAGEKWDNFQGKIDFVDCKFTY PSRPDSQVLNGLSVSISPGQTLAFVGSSGCGKSTSIQLLERFY DPDQGKVMIDGHDSKKVNVQFLRSNIGIVSQEPVLFACSIM DNIKYGDNTKEIPMERVIAAAKQAQLHDFVMSLPEKYETNV GSQGSQLSRGEKQRIAIARAIVRDPKILLLDEATSALDTESEK TVQVALDKAREGRTCIVIAHRLSTIQNADIIAVMAQGVVIEK GTHEELMAQKGAYYKLVTTGSPIS Streptococcus MEGRTVFVIAHRLSTIVNSDVILVMDHGRIIKRGDHDTLMEQ thermophilus GGTYYRLYTGSLEID Msba subfamily ABC transporter ATP-binding protein STH8232_1633 SEQ ID NO: 116 Nostoc spp. ATGTGGGGGAAACAAAGACAAAGAATCGCCATTGCACGA As11293 ABC GGGGGTTTTAAGAATTTGCAGGTTTTGATTTTAGATAAAG transporter CAACCTCGGCATTGGATAATAAAACAGAAGCAGCTATTG gene AGCGATCGCTGGTGTTGACTGTTGACCAATGA SEQ ID NO: 117 Nostoc spp. MWGKQRQRIAIARGGFKNLQVLILDKATSALDNKTEAMER As11293 ABC SINLTVDQ. transporter protein SEQ ID NO: 118 Neisseria ATGAATATCCTCAGTAAAATCAGCAGCTTTATCGGAAAA meningitides ACATTTTCCCTCTGGGCCGCGCTCTTTGCCGCCGCCGCTTT (MC58) TTTCGCGCCCGACACCTTCAAATGGGCGGGGCCTTATATT  ASBTNN4 bile CCTTGGCTGTTGGGCATTATTATGTTCGGTATGGGTTTGA acid sodium CGCTCAAACCTTCCGACTTCGATATTTTGTTCAAACATCC symporter CAAAGTCGTCATCATCGGCGTAATCGCACAATTCGCCATT (NMB0705) ATGCCGGCAACCGCCTGGCTGCTGTCCAAACTGTTGAACC SEQ ID NO: TGCCTGCCGAAATCGCGGTCGGCGTGATTTTGGTCGGCTG 119 CTGCCCGGGCGGTACGGCTTCCAATGTGATGACCTATCTG GCGCGTGGCAATGTGGCTTTGTCGGTTGCCGTTACGTCTG TTTCCACCCTGATTTCCCCATTGCTGCTCCCGCCATCTTC TTTCCACCCTGATTTCCCCATTGCTGACTCCCGCCATCTTC CTGATGCTTGCCGGCGAAATGCTGGAAATCCAAGCGGCC GGTATGTTGATGTCCATCGTCAAAATGGTTTTGCTCCCCA TTGTTTTGGGTTTGATTGTCCATAAGGTTTTGGGCAGTAA AACCGAAAAGCTGACCGATGCGCTGCCGCTGGTTTCCGTT GCCGCCATCGTGCTGATTATCGGCGCGGTTGTTGGGGCAA GCAAAGGCAAGATTATGGAAAGCGGCCTTGCTGATTTTTG CGGTTGTCGTACTCCACAACGGCATCGGCTACCTGCTCGG CTTCTTTGCCGCCAAATGGACCGGCCTGCCTTATGATGCA CAAAAAACGCTGACCATCGAAGTCGGTATGCAAAACTCG GGCCTGGCCGCCGCGCTTGCCGCCGCACACTTTGCCGCCG CGCCGGTCGTTGCCGTTCCGGGCGCATTGTTCAGCGTGTG GCACAATATCTCCGGCTCGCTGCTGGCAACTTATTGGGCG GCCAAAGCCGGTAAACATAAAAAACCCTAA Neisseria MNILSKISSFIGKTFSLWAALFAAAAFFAPDTFKWAGPYIPW meningitides LLGIIMFGMGLTLKPSDFDILFKHPKVVIIGVIAQFAIMPATA (MC58) WLLSKLLNLPAEIAVGVILVGCCPGGTASNVMTYLARGNVA ASBTNm bile LSVAVTSVSTLISPLLTPAIFLMLAGEMLEIQAAGMLMSIVK acid sodium MVLLPIVLGLIVHKVLGSKTEKLTDALPLVSVAAIVLIIGAVV symporter GASKGKIMESGLLIFAVVVLHNGIGYLLGFFAAKWTGLPYD protein AQKTLTIEVGMQNSGLAAALAAAHFAAAPVVAVPGALFSV SEQ ID NO: WHNISGSLLATYWAAKAGKHKKPGSENLYFQ 120

In one embodiment, the bile salt transporter is the bile salt importer CbsT1. In one embodiment, the cbsT1 gene has at least about 80% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 90% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 95% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 110. In another embodiment, the cbsT1 gene comprises the sequence of SEQ ID NO: 110. In yet another embodiment the cbsT1 gene consists of the sequence of SEQ ID NO: 110.

In one embodiment, the bile salt transporter is the bile salt importer CbsT2. In one embodiment, the cbsT2 gene has at least about 80% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 90% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 95% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 112. In another embodiment, the cbsT2 gene comprises the sequence of SEQ ID NO: 112. In yet another embodiment the cbsT2 gene consists of the sequence of SEQ ID NO: 112.

In one embodiment, the bile acid transporter is the bile acid sodium symporter ASBT_(NM). In one embodiment, the NMB0705 gene of Neisseria meningitides has at least about 80% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 90% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 95% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 117. In another embodiment, the NMB0705 gene comprises the sequence of SEQ ID NO: 117. In yet another embodiment the NMB0705 gene consists of the sequence of SEQ ID NO: 117.

In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120.

In some embodiments, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a transporter of a bile salt. In some embodiments, the heterologous gene encoding a transporter of the bile salt is operably linked to the first promoter. In other embodiments, the heterologous gene encoding a transporter of the bile salt is operably linked to a second promoter. In one embodiment, the gene encoding a transporter of the bile salt is directly operably linked to the second promoter. In another embodiment, the gene encoding a transporter of the bile salt is indirectly operably linked to the second promoter.

In some embodiments, expression of a gene encoding a transporter of a bile salt is controlled by a different promoter than the promoter that controls expression of the gene encoding the bile salt hydrolase enzyme. In some embodiments, expression of the gene encoding a transporter of a bile salt is controlled by the same promoter that controls expression of the bile salt hydrolase enzyme. In some embodiments, a gene encoding a transporter of a bile salt and the bile salt hydrolase enzyme are divergently transcribed from a promoter region. In some embodiments, expression of each of genes encoding the gene encoding a transporter of a bile salt and the gene encoding the bile salt hydrolase enzyme is controlled by different promoters.

In one embodiment, the gene encoding a transporter of a bile salt is not operably linked with its natural promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by its native promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by an inducible promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by a promoter that is stronger than its native promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by a constitutive promoter.

In another embodiment, the promoter is an inducible promoter. Inducible promoters are described in more detail infra.

In one embodiment, the gene encoding a transporter of a bile salt is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell, and a copy of a gene encoding a transporter of a bile salt from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located on a plasmid in the bacterial cell, and a copy of a gene encoding a transporter of a bile salt from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell, and a copy of the gene encoding a transporter of a bile salt from a different species of bacteria is located in the chromosome of the bacterial cell.

In some embodiments, the at least one native gene encoding the transporter of a bile salt in the bacterial cell is not modified, and one or more additional copies of the native transporter of a bile salt are inserted into the genome. In one embodiment, the one or more additional copies of the native transporter that is inserted into the genome are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the bile salt hydrolase enzyme, or a constitutive promoter. In alternate embodiments, the at least one native gene encoding the transporter is not modified, and one or more additional copies of the transporter from a different bacterial species is inserted into the genome of the bacterial cell. In one embodiment, the one or more additional copies of the transporter inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the gene encoding the bile salt hydrolase enzyme, or a constitutive promoter.

In one embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import 10% more bile salt into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more bile salt into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import two-fold more bile salt into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more bile salt into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.

Exporters of Bile Salts

The export of bile salts is mediated by proteins well known to those of skill in the art. For example, the ATP-binding cassette, sub-family B member 11 (ABCB11, also called BSEP or “bile salt export pump”) is responsible for the export of taurochoate and other cholate conjugates from hepatocytes to the bile in mammals, and mutations in this gene have been associated with progressive familial intrahepatic cholestasis type 2 (PFIC2) and hepatocellular carcinoma (see Strautnieks et al., Nature Genetics, 20(3):233-238, 1998; Knisely et al., Hepatology, 44(2):478-486, 2006; and Ho et al., Pharmacogenet. Genomics, 20(1):45-57, 2010; SEQ ID NO: 113 and SEQ ID NO:114). In bacteria, Streptococcus thermophilus comprises a bile salt export pump (Msba subfamily ABC transporter ATP-binding protein; accession F8LYG6; SEQ ID NO: 116), and Nostoc spp. are known to comprise a bile salt export pump (As11293; accession Q8YXC2; SEQ ID NO: 117 and SEQ ID NO: 118). Multiple other bile salt exporters are known in the art.

Thus, in one embodiment of the invention, when the recombinant bacterial cell comprises an endogenous bile salt exporter gene, the recombinant bacterial cells may comprise a genetic modification that reduces export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cell comprises a genetic modification that reduces export of one or more bile salts from the bacterial cell and a heterologous gene encoding a bile salt catabolism enzyme. When the recombinant bacterial cells comprise a genetic modification that reduces export of a bile salt, the bacterial cells retain more bile salts in the bacterial cell than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the recombinant bacteria comprising a genetic modification that reduces export of a bile salt may be used to retain more bile salts in the bacterial cell so that any bile salt catabolism enzyme expressed in the organism can catabolize the bile salt(s) to treat diseases associated with bile salts, including cardiovascular disease. In one embodiment, the recombinant bacteria further comprise a heterologous gene encoding a transporter of one or more bile salts.

In one embodiment, the recombinant bacterial cell comprises a genetic modification in a gene encoding a bile salt exporter wherein said bile salt exporter comprises an amino acid sequence that has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of a polypeptide encoded by a bile salt exporter gene disclosed herein. In one embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 115. In another embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 117.

In one embodiment, the genetic modification reduces export of a bile salt from the bacterial cell. In one embodiment, the bacterial cell is from a bacterial genus or species that includes but is not limited to, Streptococcus thermophilus or Nostoc spp.

In one embodiment, the genetic modification is a mutation in an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in an exporter having reduced activity as compared to a wild-type exporter protein. In one embodiment, the activity of the exporter is reduced at least 50%, at least 75%, or at least 100%. In another embodiment, the activity of the exporter is reduced at least two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation results in an exporter having no activity, i.e., results in an exporter which cannot export one or more bile salts from the bacterial cell.

It is routine for one of ordinary skill in the art to make mutations in a gene of interest. Mutations include substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide of the exporter of an amino acid. Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. No. 7,783,428; U.S. Pat. No. 6,586,182; U.S. Pat. No. 6,117,679; and Ling, et al., 1999, “Approaches to DNA mutagenesis: an overview,” Anal. Biochem., 254(2):157-78; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462; Carter, 1986, “Site-directed mutagenesis,” Biochem. J., 237:1-7; and Minshull, et al., 1999, “Protein evolution by molecular breeding,” Current Opinion in Chemical Biology, 3:284-290. For example, the lambda red system can be used to knock-out genes in E. coli (see, for example, Datta et al., Gene, 379:109-115 (2006)).

The term “inactivated” as applied to a gene refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein). The term “inactivated” encompasses complete or partial inactivation, suppression, deletion, interruption, blockage, promoter alterations, antisense RNA, dsRNA, or down-regulation of a gene. This can be accomplished, for example, by gene “knockout,” inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A deletion may encompass all or part of a gene's coding sequence. The term “knockout” refers to the deletion of most (at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) or all (100%) of the coding sequence of a gene. In some embodiments, any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome.

Assays for testing the activity of an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, export of one or more bile salts may be determined using the methods described by Telbisz and Homolya, Expert Opinion Ther. Targets, 1-14, 2015, the entire contents of which are expressly incorporated herein by reference.

In another embodiment, the genetic modification is a mutation in a promoter of an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in decreased expression of the exporter gene. In one embodiment, exporter gene expression is reduced by about 50%, 75%, or 100%. In another embodiment, exporter gene expression is reduced about two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation completely inhibits expression of the exporter gene.

Assays for testing the level of expression of a gene, such as an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, reverse-transcriptase polymerase chain reaction may be used to detect the level of mRNA expression of a gene. Alternatively, Western blots using antibodies directed against a protein may be used to determine the level of expression of the protein.

In another embodiment, the genetic modification is an overexpression of a repressor of an exporter of one or more bile salts. In one embodiment, the overexpression of the repressor of the exporter is caused by a mutation which renders the promoter of the repressor constitutively active. In another embodiment, the overexpression of the repressor of the exporter is caused by the insertion of an inducible promoter in front of the repressor so that the expression of the repressor can be induced. Inducible promoters are described in more detail herein.

In one embodiment, the recombinant bacterial cells described herein comprise at least one genetic modification that reduces export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise two genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise three genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise four genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise five genetic modifications that reduce export of one or more bile salts from the bacterial cell. GLP-2

In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-2 or proglucagon. Glucagon-like peptide 2 (GLP-2) is produced by intestinal endocrine cells and stimulates intestinal growth and enhances gut barrier function (Yazbeck et al., 2009). Obesity is associated with systemic inflammation and intestinal permeability, and commensal bacteria that produce GLP-2 may ameliorate those symptoms of the metabolic disease (Musso et al., 2010). The genetically engineered bacteria may comprise any suitable gene encoding GLP-2 or proglucagon, e.g., human GLP-2 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-2 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-2 analog, e.g., Teduglutide (Yazbeck et al., 2009). In some embodiments, the gene encoding GLP-2 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-2 production, and/or increase gut barrier enhancing potency. In some embodiments, the genetically engineered bacteria are capable of expressing GLP-2 or proglucagon in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 121 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 121 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 in low-oxygen conditions.

TABLE 12A SEQ ID NO: 121 GLP-2 SEQ ID NO: 121 HADGSFSDEMNTILDNLAARDFINWLIQTKITD

In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 analogs, including but not limited to, Gattex and teduglutide. Teduglutide is a protease resistan analog of GLP-2. It is made up of 33 amino acids and differs from GLP-2 by one amino acid (alanine is substituted by glycine). The significance of this substitution is that teduglutide is longer acting than endogenous GLP-2 as it is more resistant to proteolysis from dipeptidyl peptidase-4.

TABLE 12B SEQ ID NO: 122 Teduglutide SEQ ID NO: 122 HGDGSFSDEMNTILDNLAARDFINWLIQTKITD

In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 122 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 122 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide in low-oxygen conditions. In any of these embodiments the gene sequence encoding GLP-2 or GLP-2 analog may be operably linked to any of the indicuible promoters described herein. In any of these embodiments, the gene sequence encoding GLP-2 or GLP-2 analog may be operably linked to apromoter that it is not naturallyt linked to in nature.

Tryptophan and Metabolites

1-Tryptophan (TRP) is one of the nine essential amino acids and is the least abundant of all 21 dietary amino acids in human beings. Dietary TRP is transported from the digestive tract through the portal vein to the liver where it is used for the synthesis of proteins. The distinguishing structural characteristic of TRP is that it contains an indole functional group. Apart from protein synthesis, TRP is used in the generation of products such as serotonin, melatonin, tryptamine, indole and other indole metabolites, and kynurenine pathway metabolites (KP, collectively called the kynurenines). TRP and its catabolites have well characterized immunosuppressive and disease tolerance functions, and contribute to immune privileged sites such as eyes, brain, placenta, and testes. The kynurenine pathway represents >95% of TRP- catabolizing pathways and is now established as a key regulator of innate and adaptive immunity through its involvement in cancer, autoimmunity, infection, and gastrointestinal health and gut barrier integrity, and other inflammatory metabolic disorders.

Several KP Pathway metabolites, most notably kynurenine, have been shown to be activating ligands for the arylcarbon receptor (AhR; also known as dioxin receptor). Kynurenine (KYN) was initially shown in the cancer setting as an endogenous AHR ligand in immune and tumor cells, acting both in an autocrine and paracrine manner, and promoting tumor cell survival.

In the gut, the kynurenine pathway metabolism is regulated by gut microbiota, which can regulate tryptophan availability for kynurenine pathway metabolism. Tryptophan may be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and converted to kynurenine, where it functions in the suppression of T cell responses and promotion of Treg cells.

More recently, additional tryptophan metabolites, collectively termed “indoles”, herein, also have been shown to function as AhR agonists. The metabolites include for example, indole-3 aldehyde, indole-3 acetate, indole-3 propionic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ, etc., and tryptamine (are, see e.g., Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 35A and FIG. 35B and elsewhere herein, and Lama et al., Nat Med. 2016 Jun; 22(6):598-605; CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands). The majority of these metabolites are generated by the microbiota; some are generated by the human host and/or taken up from the diet.

Ahr best known as a receptor for xenobiotics such as polycyclic aromatic hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in addition to kynurenine, other tryptophan metabolites, e.g., indoles (described in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 32 and elsewhere herein, tryptamine, and kynurenic acide (KYNA) have recently been identified as endogenous AhR ligands mediating immunosuppressive functions. To induce transcription of AhR target genes in the nucleus, AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF-KB subunit RelB. Studies on human cancer cells have shown that KYN activates the AhR-ARNT associated transcription of IL-6, which induced autocrine activation of IDO1 via STAT3. This AhR-IL-6-STAT3 loop is associated with a poor prognosis in lung cancer, supporting the idea that IDO/kynurenine-mediated immunosuppression enables the immune escape of tumor cells.

More recently, some indole metabolites, including but not limited to indole 3 propionic acid, have been shown to exert their effect through Pregnane X receptor (PXR), which also thought to play a key role as an essential regulator of intestinal barrier function.

Kynurenine Pathway Kynurenine, IDO, and TDO

The rate-limiting conversion of tryptophan to kynurenine (KYN) may be mediated by either of two forms of indoleamine 2, 3-dioxygenase, IDO1 expressed ubiquitously, IDO2 expressed in kidneys, epididymis, testis, and liver or by tryptophan 2,3-dioxygenase (TDO) expressed in the liver and brain.

The tryptophan kynurenine pathway is also expressed in a large number of microbiota, most prominently in Enterobacteriaceae, and kynurenine and metabolites may be synthesized in the gut (Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91). In some embodiments, the genetically engineered bacteria comprise one or more heterologous bacterially derived genes from Enterobacteriaceae, e.g. whose gene products catalyze the conversion of TRP:KYN.

In one embodiment, the genetically engineered bacteria comprise any suitable gene or genes for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise one or more of the following: a gene or gene cassette for producing a tryptophan transporter, a gene or gene cassette for producing IDO-1, and a gene or gene cassette for producing TDO. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions. In some embodiments the genetically engineered bacteria secrete an enzyme which produces kynurenine.

Post-Kynurenine Metabolism

As shown in FIG. 32, kynurenine is further metabolized along the two distinct routes competing for kynurenine as a substrate: (a) KYN, kynurenic acid (KYNA) pathway; and (b) KYN, nicotinamide adenine dinucleotide (NAD) pathway.

Kynurenic Acid, Xanthurenic Acid, Anthranillic Acid

Kynurenine is further metabolized along the two distinct routes competing for KYN as a substrate: (a) KYN, kynurenic acid (KYNA) pathway; and (b) KYN, nicotinamide adenine dinucleotide (NAD) pathway. Along one arm, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA). KYNA is generated by kynurenine aminotransferases (KAT I, II, III) and can also bind AHR and GPCRs, e.g., GPR35, glutamate receptors, N-methyl D-aspartate (NMDA)- receptors.

The major nerve supply to the gut is also activated the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, increasing or decreasing kynurenic acid levels may be beneficial to optimally regulate gut motility or gut inflammation.

KYNA also has signaling functions through activation of its recently identified receptor, GPR35. GPR35 is predominantly detected in immune cells in the gastrointestinal tract, and might be involved in nociceptive perception. KYNA might have an anti-inflammatory effect by inhibition of lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha secretion in peripheral blood mononuclear cells.

Increased concentrations of KYNA and xanthurenic acid (3-Hydroxy KYNA, XA) were detected in the plasma of patients with type 2 diabetes, presumably due to chronic stress or the low-grade inflammation that are prominent risk factors for diabetes. Thermochemical and kinetic data show that KYNA and XA are the best free- radical scavengers from the eight tested TRP metabolites, suggesting that the production is a regulatory mechanism to attenuate damage by the inflammation-induced production of reactive oxygen species, e.g., in type two diabetes.

The genetically engineered bacteria may comprise any suitable gene or genes for producing kynurenic acid. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid, e.g., from kynurenine through a circuit comprising gene(s) or gene sequence(s) compring kynurenine--oxoglutarate transaminase or an equivalent thereof. In some embodiments, the genetically engineered bacteria comprising gene(s) or gene sequence(s) encoding kynurenine--oxoglutarate transaminase.

In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions. In some embodiments, the genetically engineered bacteria secrete an enzyme for the production of kynurenic acid.

In other embodiments, the genetically engineered bacteria are capable of reducing levels of kynurenic acid, e.g., though overexpression of enzymes catabolizing kynurenic acid described herein.

The KYN-nicotinamide adenine dinucleotide Pathway

The major enzymes of the KYN-NAD pathway are KYN-3-monooxygenase and kynureninase. Among more than 30 intermediate metabolites (collectively named “kynurenines”) are NMDA agonists (quinolinic and picolinic acids) and free radical generators (3-hydroxykynurenine and 3-hydroxyanthranilic acids). One metabolite, xanthurenic acid, reacts with insulin with formation of a complex indistinguishable from insulin. Quinolinic acid (a glutamate receptor agonist) and picolinic acids stimulate inducible nitric oxide synthase (iNOS and together with 3- hydroxykynurenine and 3-hydroxyanthranilic acids might increase lipid peroxidation, and trigger an arachidonic acid cascade resulting in the increased production of inflammatory factors. As such a means to downregulate such KP metabolites is beneficial, e.g., in the treatment of inflammatory metablic diseases, e.g., T2DM and others described herein.

Further, Anthranilic and xanthurenic acid can act as antioxidants in certain chemical environments.

Therefore, finding a means to upregulate and/or downregulate the levels of flux through the KP and to reset relative amounts and/or ratios of tryptophan and its various bioactive metabolites may be useful in the prevention, treatment and/or management of metablic diseases as described herein. The present disclosure describes compositions for modulating, regulating and fine tuning tryptophan and tryptophan metabolite levels, e.g., KP metabolite levels, e.g., in the serum or in the gastrointestinal system, through genetically engineered bacteria which comprise circuitry enabling the synthesis, bacterial uptake and catabolism of tryptophan and/or tryptophan metabolites, e.g., KP metabolites, and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases.

In certain embodiments, the genetically engineered bacteria comprise one or more genes(s) or gene cassettes, which can synthesize tryptophan and/or one or more of its metabolites, e.g., KP metablites, thereby modulating local and/or systemic concentrations and or ratios of tryptophan and/or one or more of its metabolites.

In some embodiments, the genetically engineered bacteria modulate the inflammatory status, influence immunosuppression, disease tolerance, gut barrier function, satiety.

Other Indole Tryptophan Metabolites

In addition to kynurenine and KYNA, numerous compounds have been proposed as endogenous AHR ligands, many of which are generated through pathways involved in the metabolism of tryptophan and indole (Bittinger et al., 2003; Chung and Gadupudi, 2011) A large number of metabolites generated through the tryptophan indole pathway are generated by microbiota in the gut. For example, bacteria take up tryptophan, which can be converted to mono-substituted indole compounds, such as indole acetic acid (IAA) and tryptamine, and other compounds, which have been found to activate the AHR (Hubbard et al., 2015, Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles; Nature Scientific Reoports 5:12689).

In the gastronintestinal tract, diet derived and bacterially AhR ligands promote IL-22 production by innate lymphoid cells, referred to as group 3 ILCs (Spits et al., 2013, Zelante et al., Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22; Immunity 39, 372-385, August 22, 2013). AHR is essential for IL-22-production in the intestinal lamina propria (Lee et al., Nature Immunology 13, 144-151 (2012); AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch).

Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of IL-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.

Additionally, indole metabolites have been suggested to be beneficial in the treatment of metabolic disease, such as type2 diabetes. For example, in addition to its enhancement of the gut barrier function, indole has been found to promote GLP-1 secretion by intestinal enteroendocrine cells, i.e, indole inhibits voltage-gated K+channels, and changes the action potential properties of L cells, ultimately triggering GLP-1 secretion (Chimerel C, et a., (2014) Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells. Cell Rep 9:1202-1208).

Table 13 lists exemplary tryptophan metabolites which have been shown to bind to AhR and which can be produced by the genetically engineered bacteria of the disclosure. Thus, in some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes for the production of one or more metabolites listed in Table 13.

TABLE 13 Indole Tryptophan Metabolites Origin Compound Exogenous 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Dietary Indole-3-carbinol (I3C) Dietary Indole-3-acetonitrile (I3ACN) Dietary 3.3′-Diindolylmethane (DIM) Dietary 2-(indol-3-ylmethyl)-3.3′-diindolylmethane (Ltr-1) Dietary Indolo(3,2-b)carbazole (ICZ) Dietary 2-(1′H-indole-3′-carbony)-thiazole-4-carboxylic acid methyl ester (ITE) Microbial Indole Microbial Indole-3-acetic acid (IAA) Microbial Indole-3-aldehyde (IAId) Microbial Tryptamine Microbial 3-methyl-indole (Skatole) Yeast Tryptanthrin Microbial/Host Indigo Metabolism Microbial/Host Indirubin Metabolism Microbial/Host Indoxyl-3-sulfate (I3S) Metabolism Host Kynurenine (Kyn) Metabolism Host Kynurenic acid (KA) Metabolism Host Xanthurenic acid Metabolism Host Cinnabarinic acid (CA) Metabolism UV-Light 6-formylindolo(3,2-b)carbazole (FICZ) Oxidation Microbial metabolism

In addition, some indole metabolites may exert their effect through Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function. PXR-deficient (Nr1i2-/-) mice showed a distinctly “leaky”gut physiology coupled with upregulation of the Toll-like receptor 4 (TLR4), a receptor well known for recognizing LPS and activating the innate immune system (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, Aug. 21, 2014). In particular, indole 3-propionic acid (IPA), produced by microbiota in the gut, has been shown to be a ligand for PXR in vivo.

As a result of PXR agonism, indole metabolite levels e.g., produced by commensal bacteria, or by genetically engineered bacteria, may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. In other words, low levels of IPA and/or PXR and an excess of TLR4 may lead to intestinal barrier dysfunction, while increasing levels of IPA may promote PXR activation and TLR4 downregulation, and improved gut barrier health.

In other embodiments, IPA producing circuits comprise enzymes depicted and described in FIG. 43 and FIG. 44 and elsewhere herein. Thus, in some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more enzymes selected from TrpDH: tryptophan dehydrogenase (e.g., from from Nostoc punctiforme NIES-2108); FldH1/F1dH2: indole-3-lactate dehydrogenase (e.g., from Clostridium sporogenes); FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase (e.g., from Clostridium sporogenes); FldBC: indole-3-lactate dehydratase, (e.g., from Clostridium sporogenes); FldD: indole-3-acrylyl-CoA reductase (e.g., from Clostridium sporogenes); AcuI: acrylyl-CoA reductase (e.g., from Rhodobacter sphaeroides); 1pdC: Indole-3-pyruvate decarboxylase (e.g., from Enterobacter cloacae); 1ad1: Indole-3-acetaldehyde dehydrogenase (e.g., from Ustilago maydis); and Tdc: Tryptophan decarboxylase (e.g., from Catharanthus roseus or from Clostridium sporogenes). In some embodiments, the engineered bacteria comprise gene sequence(s) and/or gene cassette(s) for the production of one or more of the following: indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis(TrA).

Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH₃, NAD(P)H and H⁺. Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indol-3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (F1dA) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (F1dD) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate decarboxylase (1pdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA).

Although microbial degradation of tryptophan to indole-3-propionate has been shown in a number of microorganisms (see, e.g., Elsden et al., The end products of the metabolism of aromatic amino acids by Clostridia, Arch Microbiol. 1976 Apr 1; 107(3):283-8), to date, the bacterial entire biosynthetic pathway from tryptophan to IPA is unknown. In Clostridium sporogenes, tryptophan is catabolized via indole-3-pyruvate, indole-3-lactate, and indole-3-acrylate to indole-3-propionate (O′Neill and DeMoss, Tryptophan transaminase from Clostridium sporogenes, Arch Biochem Biophys. 1968 Sep 20; 127(1):361-9). Two enzymes that have been purified from C. sporogenes are tryptophan transaminase and indole-3-lactate dehydrogenase (Jean and DeMoss, Indolelactate dehydrogenase from Clostridium sporogenes, Can J Microbiol. 1968 Apr.; 14(4):429-35). Lactococcus lactis, catabolizes tryptophan by an aminotransferase to indole-3-pyruvate. In Lactobacillus casei and Lactobacillus helveticus tryptophan is also catabolized to indole-3-lactate through successive transamination and dehydrogenation (see, e.g., Tryptophan catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts Gummalla, S., Broadbent, J. R. J. Dairy Sci 82:2070-2077, and references therein).

L-tryptophan transaminase (e.g., EC 2.6.1.27, e.g., Clostridium sporogenes or Lactobacillus casei) converts L-tryptophan and 2-oxoglutarate to (indo1-3y1)pyruvate and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indol-3y1) pyruvate and NADH and H+ to indole-3 lactate and NAD+.

In some embodiments, the engineered bacteria comprise gene sequence encoding one or more enzymes selected from tryptophan transaminase (e.g., from C. sporogenes) and/or indole-3-lactate dehydrogenase (e.g., from C. sporogenes), and/or indole-3-pyruvate aminotransferase (e.g., from Lactococcus lactis). In other embodiments, such enzymes encoded by the bacteria are from Lactobacillus casei and/or Lactobacillus helveticus.

In other embodiments, the engineered bacteria comprise IPA-producing circuits comprising enzymes depicted and described in FIG. 43 and FIG. 44 and elsewhere herein. Thus, in some embodiments, the engineered bacteria comprise gene sequence encoding one or more enzymes shown in FIG. 43 and FIG.44.

Methoxyindole pathway, Serotonin and Melatonin

The methoxyindole pathway leads to formation of serotonin (5-HT) and melatonin. Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine synthesized in a two-step enzymatic reaction: First, enzymes encoded by one of two tryptophan hydroxylase genes (Tphl or Tph2) catalyze the rate-limiting conversion of tryptophan to 5-hydroxytryptophan (5-HTP). Subsequently, 5-HTP undergoes decarboxylation to serotonin.

The majority (95%-98%) of total body serotonin is found in the gut (Berger et al., 2009). Peripheral serotonin acts autonomously on many cells, tissues, and organs, including the cardiovascular, gastrointestinal, hematopoietic, and immune systems as well as bone, liver, and placenta (Amireault et al., 2013). Serotonin functions as a ligand for any of 15 membrane-bound mostly G protein-coupled serotonin receptors (5-HTRs) that are involved in various signal transduction pathways in both CNS and periphery. Intestinal serotonin is released by enterochromaffin cells and neurons and is regulated via the serotonin re-uptake transporter (SERT). The SERT is located on epithelial cells and neurons in the intestine. Gut microbiota are interconnected with serotonin signaling and are for example capable of increasing serotonin levels through host serotonin production (Jano et al., Cell. 2015 Apr 9; 161(2):264-76. doi: 10.1016/j.ce11.2015.02.047. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis).

Modulation of tryptophan metabolism, especially serotonin synthesis is considered a novel potential strategy the treatment of gastrointestinal (GI) disorders and obesity related disorders, such as type 2 diabetes. In mice that lacked the 5-1-iT2C receptor. insulin resistance and development of type 2 diabetes was observed and they later overate and became obese, and 5-HT2C receptor agonists improve blood glucose tolerance.

In some embodiments, the engineered bacteria comprise gene sequence encoding one or more tryptophan hydroxylase genes (Tph1 or Tph2). In some embodiments, the engineered bacteria further comprise gene sequence for decarboxylating 5-HTP. In some embodiments, the engineered bacteria comprise gene sequence for the production of 5-hydroxytryptophan (5-HTP). In some embodiments, the engineered bacteria comprise gene sequence for the production of seratonin.

In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the gut, e.g., decrease or increase serotonin levels, e.g, in the gut and in the circulation. In certain embodiments, the genetically engineered bacteria influence serotonin synthesis, release, and/or degradation. In some embodiments, the genetically engineered bacteria may modulate the serotonin levels in the gut to improve gut barrier function, modulate the inflammatory status, improve glucose tolerance, reduce insulin resistance or otherwise ameliorate symptoms of a metabolic disease and/or an gastrointestinal disorder or inflammatory disorder. In some embodiments, the genetically engineered bacteria take up serotonin from the environment, e.g., the gut. In some embodiments, the genetically engineered bacteria release serotonin into the environment, e.g., the gut. In some embodiments, the genetically engineered modulate or influence serotonin levels produced by the host. In some embodiments, the genetically engineered bacteria counteract microbiota which are responsible for altered serotonin function in many metabolic diseases.

In some embodiments, the genetically engineered bacteria comprise gene sequence encoding tryptophan hydroxylase (TpH (land/or2)) and/or 1-amino acid decarboxylase, e.g. for the treatment of constipation-associated metablic disorders. In some embodiments, the genetically engineered bacteria comprise genetic cassettes which allow trptophan uptake and catalysis, reducing trptophan availability for serotonin synthesis (serotonin depletion). In some embodiments, the genetically engineered bacteria comprise cassettes which promote serotonin uptake from the environment, e.g., the gut, and serotonin catalysis.

Additionally, serotonin also functions a substrate for melatonin biosynthesis. Melatonin acts as a neurohormone and is associated with the development of circadian rhythm and the sleep-wake cycle. It has been postulated that melatonin may have a role in glucose metabolism, and several lines of evidence suggest that low melatonin secretion or reduced melatonin signaling can impair insulin sensitivity and lead to type 2 diabetes. For example, Loss-of-function mutations in the melatonin receptor are associated with insulin resistance and type 2 diabetes and McMullan et al observed that lower melatonin secretion was iassociated with a higher risk of developing type 2 diabetes. (see, e.g., McMullan et al., Melatonin secretion and the incidence of type 2 diabetes JAMA. 2013 Apr. 3; 309(13): 1388-1396).

In bacteria, melatonin is synthesized indirectly with tryptophan as an intermediate product of the shikimic acid pathway. In these cells, synthesis starts with d-erythrose-4-phosphate and phosphoenolpyruvate. In some embodiments, the genetically engineered bacteria comprise an endogenous or exogenous cassette for the production of melatonin. As a non-limiting example, the cassette is described in Bochkov, Denis V.; Sysolyatin, Sergey V.; Kalashnikov, Alexander I.; Surmacheva, Irina A. (2011). “Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources”. Journal of Chemical Biology 5 (1): 5-17. doi:10.1007/s12154-011-0064-8.

In a non-limiting example, genetically engineered bacteria convert tryptophan and/or serotonin to melatonin by, e.g., tryptophan hydroxylase (TPH), hydroxyl-O-methyltransferase (HIOMT), N-acetyltransferase (NAT), and aromatic-amino acid decarboxylase (AAAD), or equivalents thereof, e.g., bacterial equivalents.

Tryptophan and Tryptophan Metabolite Circuits Decreasing Exogenous Tryptophan

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan and/or the level of a tryptophan metabolite. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. Tryptophan transporters may be expressed or modified in the recombinant bacteria described herein in order to enhance tryptophan transport into the cell. Specifically, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a tryptophan transporter which may be used to import tryptophan into the bacteria.

The uptake of tryptophan into bacterial cells is mediated by proteins well known to those of skill in the art. For example, three different tryptophan transporters, distinguishable on the basis of their affinity for tryptophan have been identified in E. coli (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17). The bacterial genes mtr, aroP, and tnaB encode tryptophan permeases responsible for tryptophan uptake in bacteria. High affinity permease, Mtr, is negatively regulated by the trp repressor and positively regulated by the TyR product (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17 and Heatwole, et al. (1991) J. Bacteriol. 173: 3601-04), while AroP is negatively regulated by the tyR product (Chye et al. (1987) J. Bacteriol. 169:386-93).

In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. In one embodiment, the at least one gene encoding a tryptophan transporter is a gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the bacterial cell described herein has been genetically engineered to comprise at least one heterologous gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli mtr gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli aroP gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli tnaB gene.

In some embodiments, the tryptophan transporter is encoded by a tryptophan transporter gene derived from a bacterial genus or species, including but not limited to, Escherichia, Corynebacterium, Escherichia coli, Saccharomyces cerevisiae or Corynebacterium glutamicum. In some embodiments, the bacterial species is Escherichia coli. In some embodiments, the bacterial species is Escherichia coli strain Nissle.

Assays for testing the activity of a tryptophan transporter, a functional variant of a tryptophan transporter, or a functional fragment of transporter of tryptophan are well known to one of ordinary skill in the art. For example, import of tryptophan may be determined using the methods as described in Shang et al. (2013) J. Bacteriol. 195:5334-42, the entire contents of each of which are expressly incorporated by reference herein.

In one embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 10% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import two-fold more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.

In addition to the tryptophan uptake transporters, in some embodiments, the genetically engineered bacteria further comprise a circuit for the production of tryptophan metabolites, as described herein, e.g., for the production of kynurenine, kynurenine metabolites, or indole tryptophan metabolites as shown in Table 13.

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan, e.g., in combination with the production of indole metabolites, through expression of gene(s) and gene cassette(s) described herein. In some embodiments, the gene sequences(s) are driven by an inducible promoter. In some embodiments, the gene sequences(s) are driven by a constitutive promoter.

Increasing Kynurenine

In some embodiments, the genetically engineered bacteria are capable of producing kynurenine.

In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprise one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise on or more gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenine from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 37E and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine-oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido 1 and/or tdo2 and/or bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido 1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.

In any of these embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation, and/or a metabolic disorder. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with metabolic disorders, such as liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenine, which are bacterially derived. In some embodiments, the enzymes for TRP to KYN conversion are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

In some embodiments, the one or more genes for producing kynurenine are modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein.

In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Increasing Tryptophan

In some embodiments, the genetically engineered microorganisms of the present disclosure are capable of producing tryptophan. Exemplary circuits for the production of tryptophan are shown in FIG. 36(A-D), FIG. 37A.

In some embodiments, the genetically engineered bacteria that produce tryptophan comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon. In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of E. coli . (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of B. subtilis. (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis.

Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, chorismate. Thus, in some embodiments, the genetically engineered bacteria optionally comprise sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway and one or more gene sequences encoding one or more enzymes of the chorismate biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes.

In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 10). Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD+to NADH. As part of Tryptophan biosynthesis, E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved (see, e.g., FIG. 40A and FIG. 40B, FIG. 36C, FIG. 36D.

In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 15).

In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function.

In any of these embodiments the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 15.

The inner membrane protein YddG of Escherichia coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbial Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan, under the control of a promoter that is activated by low-oxygen conditions, by inflammatory conditions, liver damage, and.or metabolic disease, such as any of the promoters activated by said conditions and described herein. In some embodiments, the genetically engineered bacteria expresses one or more genes for producing tryptophan. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Table 14 lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure.

TABLE 14 Tryptophan Synthesis Cassette Sequences Description Sequence Tet-regulated taagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataagaaggctggact Tryptophan gcaccttggtgatcaaataattcgatagatgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct operon tattagcgacttgatgacttgatatccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata SEQ ID NO: atgcattactagtgaaaaaccttgaggcataaaaaggctaattgattttcgagagtttcatactgatttctgtagg 123 ccgtgtacctaaatgtacttttgaccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa aaaatatgccagattcccatctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg tcgagcaaagcccgcttattattacatgccaatacaatgtaggctgactacacctagatctgggcgagtttacg ggttgttaaaccttcgattccgacctcattaagcagactaatgcgctgttaatcactttacttttatctaatctagaca tcattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtg aactctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccgactctcgaactgct aacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcaccagttgtgtggggatcgtccggcaacg ctgctgctggaatccgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactg gataacgccagcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcacccgcctgtca gtccactgctggatgaagacgcccgcttatgctcccatcggtattgacgctaccgcttattacagaatctgaga atgtaccgaaggaagaacgagaagcaatgacttcggcggcctgactcttatgaccagtggcgggatttgaaa atttaccgcaactgtcagcggaaaatagctgccctgatactgatttatctcgctgaaacgctgatggtgattgac catcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcactgc tcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtaccgtgccgcatat gcgagtgaatgtaaccagagcgatgaagagacggtggtgtagtgcgatgagcaaaaagcgattcgcgccg gagaaattaccaggtggtgccatctcgccgtactctctgccctgcccgtcaccgctggcagcctattacgtgct gaaaaagagtaatcccagcccgtacatgatatatgcaggataatgatttcaccctgatggcgcgtcgccggaa agacgctcaagtatgacgccaccagccgccagattgagatttacccgattgccggaacacgtccacgcggtc gtcgtgccgatggacgctggacagagacctcgacagccgcatcgaactggagatgcgtaccgatcataaag agctactgaacatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagccgc tacgtcgccgatctcaccaaagagaccgttactcttacgtgatgcacctagtctcccgcgagaggtgagctgc gccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagcggtgcaccgaaagt acgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcggcagctacggcggcgcggtaggttatat accgcgcatggcgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtgc aagccggtgctggcgtagtccagattctgaccgcagtcggaagccgacgaaactcgtaataaagcccgcgc tgtactgcgcgctattgccaccgcgcatcatgcacaggagacgactaatggctgacattctgctgctcgataat atcgactcattacgtacaacctggcagatcagagcgcagcaatggtcataacgtggtgatttaccgcaaccata accggcgcagaccttaattgaacgcctggcgacgatgagcaatccggtgctgatgctactcctggccccggt gtgccgagcgaagccggagtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatag cctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattcacacggtaaa gcgtcgagcattgaacatgacggtcaggcgatgatgccggattaacaaacccgctgccagtggcgcgttatc actcgctggaggcagtaacattccggccggataaccatcaacgcccatataatggcatggtgatggcggtgc gtcacgatgcagatcgcgtagtggattccagaccatccggaatccattcttactacccagggcgctcgcctgct ggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattctggaaaaa ctgtatcaggcacagacgcttagccaacaagaaagccaccagctgattcagcggtggtacgtggcgagctga agccggaacaactggcggcggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccgg ggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgatgccgatatcgtcggt actggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcgggctga aagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcggatctgctggcggcgacggtat taatatgatatgaacgccgataaatcgcgccaggcgctggatgagttaggcgtctgatcctattgcgccgaa gtatcacaccggattccgccatgcgatgccggacgccagcaactgaaaacccgcactctgacaacgtgctg ggaccattgattaacccggcgcatccgccgctggcgctaattggtgatatagtccggaactggtgctgccgatt gccgaaaccagcgcgtgctggggtatcaacgcgcggcagtggtgcacagcggcgggatggatgaagatc attacacgcgccgacaatcgagccgaactacatgacggcgaaattaagagctatcaattgaccgctgaagatt ttggcctgacaccctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacac gcagttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgaaatgcgcct gcatggccatgaagatctgcaagccaatgcgcaaaccgacttgaggtactgcgcagtggaccgcttacgaca gagtcaccgcactggcggcacgagggtaaatgatgcaaaccgattagcgaaaatcgtcgcagacaaggcg atttgggtagaaacccgcaaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcga cattatatgatgcacttcagggcgcacgcacggcgatattctggagtgtaaaaaagcgtcgccgtcaaaaggc gtgatccgtgatgatacgatccggcacgcattgccgccatttataaacattacgatcggcaatacagtgctgac tgatgagaaatattacaggggagattgatacctccccatcgtcagccaaatcgccccgcagccgatatatgta aagacacattatcgatccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaatgattcag tactggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagt cagtaatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgaggcatcaacaaccgcgatct gcgcgatagtcgattgatctcaaccgtacccgcgagcttgcgccgaaactggggcacaacgtgacggtaatc agcgaatccggcatcaatacttacgctcaggtgcgcgagttaagccacttcgctaacggctactgattggacg gcgttgatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcct gacacgtgggcaagatgctaaagcagatatgacgcgggcgcgatttacggtgggttgatttttgttgcgacat caccgcgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccgttgcagtatgttggcgtgtt ccgcaatcacgatattgccgatgtggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcatggt aatgaagatcagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaag tgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcggga gcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctggcggggggcttaggc gcagataactgcgtggaagcggcacaaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaac cgggtatcaaagacgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaatga caacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgcca gctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaaact atgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatctga agcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagc ggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcg ccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttccgg atgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatgagg cgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatcctta cccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagagaagg tcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatcaac gaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgcacc gttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaatt gaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcact ggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaagg gatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaaaa agagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagc acgaggggaaatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattc gttcctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtg ctgacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacact gcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccg accattcccatcggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcga gaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgc gttgcgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctctt acggtcgtggttacacctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccc tcaatcatctggttgcgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccgg atcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgag caacatattaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgc gcagttaatacgcatggcatggatgaCCGATGGTAGTGTGGGGTCTCCCCATGCG AGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGT CGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGC TCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGC GAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAA CTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCC TTTTTGCGTGGCCAGTGCCAAGCTTGCATGCGTGC Tet repressor taagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataagaaggctggctct SEQ ID gcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct NO: 124 tctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata atgcattctctagtgaaaaaccttgaggcataaaaaggctaattgattttcgagagtttcatactgatttctgtagg ccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa aaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacg ggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagaca t tetRitetA cattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtga promoters and actctagaaataattttgtttaactttaagaaggagatatacat RBS and leader region SEQ ID NO 125: trpE atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttt SEQ ID NO: tcaccagttgtgtggggatcgtccggcaacgctgctgctggaatccgcagatatcgacagcaaagatgatttaa 126 aaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgacactgtcacaatccaggcgctttcc ggcaatggagaagccctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaa actgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttt tgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcttcggcggcct gttctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaaaatagctgccctgatttctgttt ttatctcgctgaaacgctgatggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctcc gaatgaagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgc cgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggtgta gtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctcgccgtttctctctgccct gcccgtcaccgctggcagcctattacgtgctgaaaaagagtaatcccagcccgtacatgattttatgcaggata atgatttcaccctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgagattt acccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgc atcgaactggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatctcgcccgtaatgac ctggcacgcatttgcacacccggcagccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgat gcacctagtctcccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgac gcggcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcattgtgatccgctc ggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtccttgattctgttccgcagtcg gaagccgacgaaactcgtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggag acgttcta TrpE MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLESADIDSKD SEQ ID NO: DLKSLLLVDSALRITALSDTVTIQALSGNGEALLTLLDNALPAGVENE 127 QSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRLLQNLLNVPKEEREA MFFGGLFSYDLVAGFENLPQLSAENSCPDFCFYLAETLMVIDHQKKST RIQASLFAPNEEEKQRLTARLNELRQQLTEAAPPLPVVSVPHMRCECN QSDEEFGGVVRLLQKAIRAGEIFQVVPSRRFSLPCPSPLAAYYVLKKS NPSPYMFFMQDNDFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRA DGSLDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRY VADLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGTLSG APKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIVIRSALVEN GIATVQAGAGVVLDSVPQSEADETRNKARAVLRAIATAHHAQETF trpD atggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatggtc SEQ ID NO: ataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcgacgatgagcaatccg 128 gtgctgatgctttctcctggccccggtgtgccgagcgaagccggttgtatgccggaactcctcacccgcttgcg tggcaagctgccaattattggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtca ggcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaaca aacccgctgccagtggcgcgttatcactcgctggttggcagtaacattccggccggtttaaccatcaacgccca ttttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgtttgtggattccagttccatccggaatccatt cttactacccagggcgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaa cacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgttt tcagcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcatgaaaattcgcgg tgaacacccgaacgagatcgccggggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccg gattatctgtttgccgatatcgtcggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcg tttgtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcg tcggatctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgctggatgagtta ggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatgcgatgccggttcgccagcaactgaa aacccgcactctgttcaacgtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgttta tagtccggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgca cagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcgaaattaag agctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagcaattggcaggcggaacaccgg aagaaaaccgtgacattttaacacgcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcgg cgaatgtcgccatgttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggta ctgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggtaa TrpD MADILLLDNIDSFTYNLADQLRSNGHNVVIYRNHIPAQTLIERLATMS SEQ ID NO: NPVLMLSPGPGVPSEAGCMPELLTRLRGKLPIIGICLGHQAIVEAYGG 129 YVGQAGEILHGKASSIEHDGQAMFAGLTNPLPVARYHSLVGSNIPAG LTINAHFNGMVMAVRHDADRVCGFQFHPESILTTQGARLLEQTLAW AQQKLEPTNTLQPILEKLYQAQTLSQQESHQLFSAVVRGELKPEQLAA ALVSMKIRGEHPNEIAGAATALLENAAPFPRPDYLFADIVGTGGDGSN SINISTASAFVAAACGLKVAKHGNRSVSSKSGSSDLLAAFGINLDMNA DKSRQALDELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLFNVLGPLI NPAHPPLALIGVYSPELVLPIAETLRVLGYQRAAVVHSGGMDEVSLH APTIVAELHDGEIKSYQLTAEDFGLTPYHQEQLAGGTPEENRDILTRLL QGKGDAAHEAAVAANVAMLMRLHGHEDLQANAQTVLEVLRSGSA YDRVTALAARG trpC atgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagcagcaaccg SEQ ID NO: ctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttcagggcgcacgcacggc 130 gtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgc cgccatttataaacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttcct ccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctg gcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaatatcgccagcttgca gccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagtaatgaagaggaactggagcgcgccatt gcattgggggcaaaggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccg cgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggt gcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgatttgaacgccgc cgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtgggcaagatgctaaagcagcttat gacgcgggcgcgatttacggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgca ggaagtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcggaca aagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaatctgcgt gaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaactcttcccgcgcgcgattttca gcacatcgataaatatgtattcgacaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatg gtcaatcgcttggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcacaaaccg gctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctcggt tttccagacgctgcgcgcatattaa TrpC MQTVLAKIVADKAIWVETRKEQQPLASFQNEVQPSTRHFYDALQGA SEQ ID NO: RTAFILECKKASPSKGVIRDDFDPARIAAIYKHYASAISVLTDEKYFQG 131 SFDFLPIVSQIAPQPILCKDFIIDPYQIYLARYYQADACLLMLSVLDDEQ YRQLAAVAHSLEMGVLTEVSNEEELERAIALGAKVVGINNRDLRDLS IDLNRTRELAPKLGHNVTVISESGINTYAQVRELSHFANGFLIGSALM AHDDLNAAVRRVLLGENKVCGLTRGQDAKAAYDAGAIYGGLIFVAT SPRCVNVEQAQEVMAAAPLQYVGVFRNHDIADVADKAKVLSLAAV QLHGNEDQLYIDNLREALPAHVAIWKALSVGETLPARDFQHIDKYVF DNGQGGSGQRFDWSLLNGQSLGNVLLAGGLGADNCVEAAQTGCAG LDFNSAVESQPGIKDARLLASVFQTLRAY trpB atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcg SEQ ID NO: ccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaa 132 actatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatc tgaagcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcga agcggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgcca gcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttc cggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatg aggcgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatc cttacccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagaga aggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatc aacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgc accgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaa attgaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagc actggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaa gggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaa aaagagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaa gcacgaggggaaatctga TrpB MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQAQFND SEQ ID NO: LLKNYAGRPTALTKCQNITAGTNTTLYLKREDLLHGGAHKTNQVLG 133 QALLAKRMGKTEIIAETGAGQHGVASALASALLGLKCRIYMGAKDV ERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYETAH YMLGTAAGPHPYPTIVREFQRMIGEETKAQILEREGRLPDAVIACVGG GSNAIGMFADFINETDVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFG MKAPMMQTEDGQIEESYSISAGLDFPSVGPQHAYLNSTGRADYVSIT DDEALEAFKTLCLHEGIIPALESSHALAHALKMMRENPEKEQLLVVN LSGRGDKDIFTVHDILKARGEI trpA atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc SEQ ID NO: ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagtt 134 aggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcg ggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggcc ttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcga ttcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcg cacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacac ctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcg aagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccg cgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacatattaatgagc cagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgcgcagttaa TrpA MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALE SEQ ID NO: LGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGL 135 LMYANLVFNKGIDEFYAECEKVGVDSVLVADVPVEESAPFRQAALR HNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPL NHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKII EQHINEPEKMLAALKAFVQPMKAATRS

In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 14 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 14 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 14 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 14 or a functional fragment thereof.

Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 123 through SEQ ID NO: 135. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 123 through SEQ ID NO: 135. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 123 through SEQ ID NO: 135.

Table 15 depicts exemplary polypeptide sequences feedback resistant AroG and TrpE. Table 15 also depicts an exemplary TnaA (tryptophanase from E. coli ) sequence. IN some embodiments, the sequence is encoded in circuits for tryptophan catabolism to indole; in other embodimetns, the sequence is deleted from the E coli chromosome to increase levels of tryptophan.

TABLE 15 Feedback resistant AroG and TrpE and tryptophanase sequences Description Sequence Aro Gfbr: feedback MNYQNDDLRIKEIKELLPPVALLEKFPATENAANTVAHARKAI resistant 2-dehydro- HKILKGNDDRLLVVIGPCSIHDPVAAKEYATRLLTLREELQDE 3- LEIVMRVYFEKPRTTVGWKGLINDPHMDNSFQINDGLRIARK deoxyphospho- LLLDINDSGLPAAGEFLDMITLQYLADLMSWGAIGARTTESQ heptonate aldolase VHRELASGLSCPVGFKNGTDGTIKVAIDAINAAGAPHCFLSVT from E. coli KWGHSAIVNTSGNGDCHIILRGGKEPNYSAKHVAEVKEGLNK SEQ ID NO: 136 AGLPAQVMIDFSHANSSKQFKKQMDVCTDVCQQIAGGEKAII GVMVESHLVEGNQSLESGEPLAYGKSITDACIGWDDTDALLR QLASAVKARRG TrpEfbr: feedback MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLEFADI resistant DSKDDLKSLLLVDSALRITALSDTVTIQALSGNGEALLTLLDN anthranilate ALPAGVENEQSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRL synthase LQNLLNVPKEEREAMFFGGLFSYDLVAGFENLPQLSAENSCP component I from DFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNE E. coli LRQQLTEAAPPLPVVSVPHMRCECNQSDEEFGGVVRLLQKAI SEQ ID NO: 137 RAGEIFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDN DFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRD LDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVA DLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGT LSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIV IRSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRA IATAHHAQETF SerA: 2- MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL oxoglutarate DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT reductase from E. NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE coli Nissle ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE SEQ ID NO: 137 SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI HENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA DEDVAEKALQAMKAIPGTIRARLLY SerAfbr: feedback MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL resistant 2- DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT oxoglutarate NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE reductase from E. ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE coli Nissle SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE SEQ ID NO: 139 NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI AEARPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA DEDVAEKALQAMKAIPGTIRARLLY TnaA: MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE tryptophanase from DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE E. coli SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM SEQ ID NO: 140 VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKVM YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPA QALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTI PRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFT AKLKEV

In one embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. Accordingly, in one embodiment, one or more polypeptides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 136 through SEQ ID NO: 139. In another embodiment, one or more polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 136 through SEQ ID NO: 139.

In some embodiments, the endogenous TnaA polypeptide comprising SEQ ID NO: 140 is mutated or deleted.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Producing Kynurenic Acid

In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine--oxoglutarate transaminase.

In some embodiments, the gene or genes for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYNA ratio, e.g. in the circulation. In some embodiments the TRP:KYNA ratio is increased. In some embodiments, TRP:KYNA ratio is decreased.

In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenic acid, which are bacterially derived. In some embodiments, the enzymes for producing kynureic acid are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding one or more kynurenine--oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters, gene sequence(s) encoding kynureninase, and gene sequence(s) encoding one or more kynurenine--oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding kynureninase and gene sequence(s) encoding one or more kynurenine aminotransferases.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenic acid from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 37F and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with ID01. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with TDO2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine--oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 (Aspartate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 from homo sapiens.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 (Kynurenine--oxoglutarate transaminase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 from homo sapiens). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 (kynurenine--oxoglutarate transaminase 3) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cc1b1 and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3, and in combination with one or more of cc1b1 and/or cc1b2 and/or aadat and/or got2.

In any of these embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the one or more genes for producing kynurenic acid are modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Producing Indole Tryptophan Metabolites and Tryptamine

In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of indole metabolites and/or tryptamine. Exemplary circuits for the production of indole metabolites/derivatives (e.g., FIG. 34, FIG. 35) are shown in FIG. 37A through FIG. 37H, FIG. 38A through FIG. 38F, and FIG. 39A though FIG. 39C, FIG. 43, and FIG. 44.

In some embodiments, the genetically engineered bacteria comprise genetic circuitry for converting tryptophan to tryptamine. In some embodiments, the engineered bacteria comprise gene sequence encoding Tryptophan decarboxylase, e.g., from Catharanthus roseus. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetaldehyde and FICZ from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli , taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA (Monoamine oxidase, e.g., from E. coli ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetonitrile from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2, (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N- monooxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynurenine from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynureninic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g.,from homo sapiens or AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tnaA (tryptophanase, e.g., from E. coli ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate (taken up through the diet). The genetically engineered bacteria comprise a gene sequence encoding pne2 (myrosinase, e.g., from Arabidopsis thaliana). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli , taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae), iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g.,from Catharanthus roseus and/or Clostridium sporogenes), tynA (Monoamine oxidase, e.g., from E. coli ), iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli , taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 (indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IaaM (Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi), iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana, cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana), nit1 (Nitrilase, e.g., from Arabidopsis thaliana), iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.

In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. In some embodiments, the engineered bacteria produces tryptamine. Tryptophan is optionally produced from chorismate precursor, and the bacteria optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the bacteria comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine.

In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-acetate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.

In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-propionate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 36A and/or FIG. 36B and/or FIG. 36C and/or FIG. 36D. Additionally, the strain comprises a circuit as described in FIG. 44, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-3y1)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH 1 and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indo1-3-yl)pyruvate into indole-3-lactate).

In some embodiments, the engineered bacteria comprises genetic circuitry for the production of indole-3-propionic acid (IPA). In some embodiments, the engineered bacteria comprises gene sequence encoding tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus) and indole-3-acrylate reductase (e.g., from Clostridum botulinum). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-propionic acid (IPA), indole acetic acid (IAA), and/or tryptamine synthesis(TrA) circuits. In some embodiments, the engineered bacteria comprise gene sequence encoding one or more of the following: TrpDH: tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108; FldH1/F1dH2: indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole-3-lactate dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; AcuI: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. 1pdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae; lad1: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.

In some embodiments, the engineered bacteria comprise genetic circuitry for producing (indol-3-yl)pyruvate (IPyA). In some embodiments, the engineered bacteria comprise gene sequence encoing one or more of the following: tryptophan dehydrogenase (EC 1.4.1.19) (enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH₃, NAD(P)H and ft)); Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) (converts (indol-3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+); Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (F1dA) (converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA); Indole-3-acrylyl-CoA reductase (F1dD) and acrylyl-CoA reductase (Acul) (convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA); Indole-3-lactate dehydratase (FldBC) (converts indole-3-lactate-CoA to indole-3-acrylyl-CoA); Indole-3-pyruvate decarboxylase (1pdC:) (converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d)); lad1: Indole-3-acetaldehyde dehydrogenase (coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA)); Tdc: Tryptophan decarboxylase (converts tryptophan (Trp) into tryptamine (TrA)). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In any of the described embodiments, any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA; thymidine dependence).

In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 36 (A-D) and FIG. 40 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In in any of these embodiments the expression of the gene sequences for the production of the indole and other tryptophan metabolites, including, but not limited to, tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, indole, indole acetic acid FICZ, indole-3-propionic acid, is under the control of an inducible promoter. Exemplary inducible promoters which may control the expression of the biosynthetic cassettes include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite characteristic of a disorder, such as liver damage or a metabolic disease, or that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. Tryptamine

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce tryptamine from tryptophan. The monoamine alkaloid, tryptamine, is derived from the direct decarboxylation of tryptophan. Tryptophan is converted to indole-3-acetic acid (IAA) via the enzymes tryptophan monooxygenase (IaaM) and indole-3- acetamide hydrolase (IaaH), which constitute the indole-3-acetamide (IAM) pathway, see eg., FIG. 34, FIG. 35A and FIG. 35B.

A non-limiting example of such as strain is shown in FIG. 37A. Another non-limiting example of such as strain is shown in FIG. 39A. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from Clostridium sporgenenes. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s) e.g., from Ruminococcus Gnavus.

Table 15, Table 16, and Table 17 lists exemplary sequences for tryptamine production in genetically engineered bacteria.

In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Indole-3-acetaldehyde and FICZ

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetaldehyde and FICZ from tryptophan. Exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in FIG. 37B.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and ipdC.

Further exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in FIG. 37C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA.

In any of these embodiments, the genetically engineered bacteria which produce produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. Indole-3-acetic acid

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes which convert tryptophan to Indole-3-aldehyde and Indole Acetic Acid, e.g., via a tryptophan aminotransferase cassette. A non-limiting example of such a tryptophan aminotransferase expressed by the genetically engineered bacteria is in Table 14. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter, and further produce Indole-3-aldehyde and Indole Acetic Acid from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptophan and/or indole metabolite exporter.

The genetically engineered bacteria may comprise any suitable gene for producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the engineered bacteria also have enhanced export of a indole tryptophan metabolite , e.g., comprise an exporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetic acid.

Non-limiting example of such gene sequence(s) are shown in FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, and FIG. 38E, and FIG. 39B and FIG. 39E.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 from S. cerevisae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase), In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taal and/or staO and/or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taa1 and/or staO and in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1 (see, e.g., FIG. 38A).

Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 38B. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and one or more sequence(s) selected from iadl and/or aao 1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA and one or more sequence(s) selected from iadl and/or aao 1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA and one or more sequence(s) selected from iad1 and/or aao1.

Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 38C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 (indole-3-pyruvate monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 from Enterobacter cloacae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and yuc2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and yuc2.

Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 38D. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM and iaaH.

Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 38E. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 (Nitrilase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13, and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nit1 and iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13 and nit1 and iaaH.

Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 38F. Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 39E. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and/or ipdC and/or iad1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and ipdC and iad1.

In any of these embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Indole Acetic Acid and under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. Indole-3-acetonitrile

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetonitrile from tryptophan. A non-limiting example of such gene sequence(s) which allow in which the genetically engineered bacteria to produce indole-3-acetonitrile from tryptophan is depicted in FIG. 16D.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13.

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13.

In any of these embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

In some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Indole-3-Propionic Acid (IPA)

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-propionic acid from tryptophan. FIG. 43 and FIG. 44, and FIG. 39C depict schematics of exemplary circuits for the production of indole-3-propionic acid.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase from Rubrivivax benzoatilyticus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole-3-acrylate reductase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole-3-acrylate reductase from Clostridum botulinum. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a tryptophan ammonia lyase and an indole-3-acrylate reductase. In some embodiments, the indole-3-propionate-producing strain optionally produces tryptophan from a chorismate precursor, and the strain optionally comprises additional circuits for tryptophan production and/or tryptophan uptake/transport s described herein.

The genetically engineered bacteria comprise a circuit, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-3yl)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or Acul: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH1 and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3-yl)pyruvate into indole-3-lactate) (see, e.g., FIG. 44).

Another embodiment of the IPA producing strain is shown in FIG. 43.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH from Nostoc punctiforme NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC (indole-3-lactate dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD (indole-3-acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding AcuI (acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding Acul from Rhodobacter sphaeroides. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 (3-lactate dehydrogenase 1). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 (indole-3-lactate dehydrogenase 2). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 from Clostridium sporogenes). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acuI and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acuI and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acuI and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acuI and fldH2.

In any of these embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 different tryptophan metabolites. In certain embodiments the bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites selected from tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, kynurenine, kynurenic acid, indole, indole acetic acid FICZ, indole-3-propionic acid.

In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acidand/or Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Indole

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 37G and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA (tryptophanase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA from E. coli.

In any of these embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-acetonitrile under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Other Indole Metabolites

In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. Non-limiting example of such gene sequence(s) are shown FIG. 37H and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 (myrosinase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 from Arabidopsis thaliana.

In any of these embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 36, FIG. 40A and/or FIG. 40B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

In some embodiments, the genetically engineered bacteria are capable of producing these metabolites under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Tryptophan Catabolic Pathway Enzymes

Table 16A and Table 16B comprise polypeptide and polynucleotide sequences of such enzymes which are encoded by the genetically engineered bacteria of the disclosure.

TABLE 16A Tryptophan Pathway Catabolic Enzymes Description Sequence TDC: Tryptophan MGSIDSTNVAMSNSPVGEFKPLEAEEFRKQAHRMVDFIADYY decarboxylase from KNVETYPVLSEVEPGYLRKRIPETAPYLPEPLDDIMKDIQKDII Catharanthus roseus PGMTNWMSPNFYAFFPATVSSAAFLGEMLSTALNSVGFTWV SEQ ID NO: 141 SSPAATELEMIVMDWLAQILKLPKSFMFSGTGGGVIQNTTSES ILCTIIAARERALEKLGPDSIGKLVCYGSDQTHTMFPKTCKLA GIYPNNIRLIPTTVETDFGISPQVLRKMVEDDVAAGYVPLFLC ATLGTTSTTATDPVDSLSEIANEFGIWIHVDAAYAGSACICPEF RHYLDGIERVDSLSLSPHKWLLAYLDCTCLWVKQPHLLLRAL TTNPEYLKNKQSDLDKVVDFKNWQIATGRKFRSLKLWLILRS YGVVNLQSHIRSDVAMGKMFEEWVRSDSRFEIVVPRNFSLVC FRLKPDVSSLHVEEVNKKLLDMLNSTGRVYMTHTIVGGIYML RLAVGSSLTEEHHVRRVWDLIQKLTDDLLKEA TDC: Tryptophan MKFWRKYTQQEMDEKITESLEKTLNYDNTKTIGIPGTKLDDT decarboxylase from VFYDDHSFVKHSPYLRTFIQNPNHIGCHTYDKADILFGGTFDIE Clostridium RELIQLLAIDVLNGNDEEFDGYVTQGGTEANIQAMWVYRNY sporogenes FKKERKAKHEEIAIITSADTHYSAYKGSDLLNIDIIKVPVDFYS SEQ ID NO: 142 RKIQENTLDSIVKEAKEIGKKYFIVISNMGTTMFGSVDDPDLY ANIFDKYNLEYKIHVDGAFGGFIYPIDNKECKTDFSNKNVSSIT LDGHKMLQAPYGTGIFVSRKNLIHNTLTKEATYIENLDVTLSG SRSGSNAVAIWMVLASYGPYGWMEKINKLRNRTKWLCKQL NDMRIKYYKEDSMNIVTIEEQYVNKEIAEKYFLVPEVHNPTN NWYKIVVMEHVELDILNSLVYDLRKFNKEHLKAM TYNA: Monoamine MGSPSLYSARKTTLALAVALSFAWQAPVFAHGGEAHMVPM oxidase from E. coli DKTLKEFGADVQWDDYAQLFTLIKDGAYVKVKPGAQTAIVN SEQ ID NO: 143 GQPLALQVPVVMKDNKAWVSDTFINDVFQSGLDQTFQVEKR PHPLNALTADEIKQAVEIVKASADFKPNTRFTEISLLPPDKEAV WAFALENKPVDQPRKADVIMLDGKHIIEAVVDLQNNKLLSW QPIKDAHGMVLLDDFASVQNIINNSEEFAAAVKKRGITDAKK VITTPLTVGYFDGKDGLKQDARLLKVISYLDVGDGNYWAHPI ENLVAVVDLEQKKIVKIEEGPVVPVPMTARPFDGRDRVAPAV KPMQIIEPEGKNYTITGDMIHWRNWDFHLSMNSRVGPMISTV TYNDNGTKRKVMYEGSLGGMIVPYGDPDIGWYFKAYLDSGD YGMGTLTSPIARGKDAPSNAVLLNETIADYTGVPMEIPRAIAV FERYAGPEYKHQEMGQPNVSTERRELVVRWISTVGNYDYIFD WIFHENGTIGIDAGATGIEAVKGVKAKTMHDETAKDDTRYGT LIDHNIVGTTHQHIYNFRLDLDVDGENNSLVAMDPVVKPNTA GGPRTSTMQVNQYNIGNEQDAAQKFDPGTIRLLSNPNKENRM GNPVSYQIIPYAGGTHPVAKGAQFAPDEWIYHRLSFMDKQLW VTRYHPGERFPEGKYPNRSTHDTGLGQYSKDNESLDNTDAV VWMTTGTTHVARAEEWPIMPTEWVHTLLKPWNFFDETPTLG ALKKDK AAO1: Indole-3- MGEKAIDEDKVEAMKSSKTSLVFAINGQRFELELSSIDPSTTL acetaldehyde oxidase VDFLRNKTPFKSVKLGCGEGGCGACVVLLSKYDPLLEKVDEF from Arabidopsis TISSCLTLLCSIDGCSITTSDGLGNSRVGFHAVHERIAGFHATQ thaliana CGFCTPGMSVSMFSALLNADKSHPPPRSGFSNLTAVEAEKAV SEQ ID NO: 144 SGNLCRCTGYRPLVDACKSFAADVDIEDLGFNAFCKKGENRD EVLRRLPCYDHTSSHVCTFPEFLKKEIKNDMSLHSRKYRWSSP VSVSELQGLLEVENGLSVKLVAGNTSTGYYKEEKERKYERFI DIRKIPEFTMVRSDEKGVELGACVTISKAIEVLREEKNVSVLA KIATHMEKIANRFVRNTGTIGGNIMMAQRKQFPSDLATILVA AQATVKIMTSSSSQEQFTLEEFLQQPPLDAKSLLLSLEIPSWHS AKKNGSSEDSILLFETYRAAPRPLGNALAFLNAAFSAEVTEAL DGIVVNDCQLVFGAYGTKHAHRAKKVEEFLTGKVISDEVLM EAISLLKDEIVPDKGTSNPGYRSSLAVTFLFEFFGSLTKKNAKT TNGWLNGGCKEIGFDQNVESLKPEAMLSSAQQIVENQEHSPV GKGITKAGACLQASGEAVYVDDIPAPENCLYGAFIYSTMPLA RIKGIRFKQNRVPEGVLGIITYKDIPKGGQNIGTNGFFTSDLLF AEEVTHCAGQIIAFLVADSQKHADIAANLVVIDYDTKDLKPPI LSLEEAVENFSLFEVPPPLRGYPVGDITKGMDEAEHKILGSMS FGSQYFFYMETQTALAVPDEDNCMVVYSSTQTPEFVHQTIAG CLGVPENNVRVITRRVGGGFGGKAVKSMPVAAACALAASK MQRPVRTYVNRKTDMITTGGRHPMKVTYSVGFKSNGKITAL DVEVLLDAGLTEDISPLMPKGIQGALMKYDWGALSFNVKVC KTNTVSRTALRAPGDVQGSYIGEAIIEKVASYLSVDVDEIRKV NLHTYESLRLFHSAKAGEFSEYTLPLLWDRIDEFSGFNKRRKV VEEFNASNKWRKRGISRVPAVYAVNMRSTPGRVSVLGDGSIV VEVQGIEIGQGLWTKVKQMAAYSLGLIQCGTTSDELLKKIRVI QSDTLSMVQGSMTAGSTTSEASSEAVRICCDGLVERLLPVKT ALVEQTGGPVTWDSLISQAYQQSINMSVSSKYMPDSTGEYLN YGIAASEVEVNVLTGETTILRTDIIYDCGKSLNPAVDLGQIEGA FVQGLGFFMLEEFLMNSDGLVVTDSTWTYKIPTVDTIPRQFN VEILNSGQHKNRVLSSKASGEPPLLLAASVHCAVRAAVKEAR KQILSWNSNKQGTDMYFELPVPATMPIVKEFCGLDVVEKYLE WKIQQRKNV ARO9: L-tryptophan MTAGSAPPVDYTSLKKNFQPFLSRRVENRSLKSFWDASDISD amino transferase DVIELAGGMPNERFFPIESMDLKISKVPFNDNPKWHNSFTTAH from S. cerevisae LDLGSPSELPIARSFQYAETKGLPPLLHFVKDFVSRINRPAFSD SEQ ID NO: 145 ETESNWDVILSGGSNDSMFKVFETICDESTTVMIEEFTFTPAM SNVEATGAKVIPIKMNLTFDRESQGIDVEYLTQLLDNWSTGP YKDLNKPRVLYTIATGQNPTGMSVPQWKREKIYQLAQRHDF LIVEDDPYGYLYFPSYNPQEPLENPYHSSDLTTERYLNDFLMK SFLTLDTDARVIRLETFSKIFAPGLRLSFIVANKFLLQKILDLAD ITTRAPSGTSQAIVYSTIKAMAESNLSSSLSMKEAMFEGWIRW IMQIASKYNHRKNLTLKALYETESYQAGQFTVMEPSAGMFIII KINWGNFDRPDDLPQQMDILDKFLLKNGVKVVLGYKMAVCP NYSKQNSDFLRLTIAYARDDDQLIEASKRIGSGIKEFFDNYKS aspC: aspartate MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKT amino transferase PVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKG from E. coli SALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSN SEQ ID NO: 146 PSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEA QAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLF DFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYN ERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASV VATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGAN RDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVA GMTPDNMAPLCEAIVAVL TAA1: L-tryptophan- MVKLENSRKPEKISNKNIPMSDFVVNLDHGDPTAYEEYWRK pyruvate MGDRCTVTIRGCDLMSYFSDMTNLCWFLEPELEDAIKDLHGV amino transferase VGNAATEDRYIVVGTGSTQLCQAAVHALSSLARSQPVSVVA from Arabidopsis AAPFYSTYVEETTYVRSGMYKWEGDAWGFDKKGPYIELVTS thaliana PNNPDGTIRETVVNRPDDDEAKVIHDFAYYWPHYTPITRRQD SEQ ID NO: 147 HDIMLFTFSKITGHAGSRIGWALVKDKEVAKKMVEYIIVNSIG VSKESQVRTAKILNVLKETCKSESESENFFKYGREMMKNRWE KLREVVKESDAFTLPKYPEAFCNYFGKSLESYPAFAWLGTKE ETDLVSELRRHKVMSRAGERCGSDKKHVRVSMLSREDVFNV FLERLANMKLIKSIDL STAO: L-tryptophan MTAPLQDSDGPDDAIGGPKQVTVIGAGIAGLVTAYELERLGH oxidase from HVQIIEGSDDIGGRIHTHRFSGAGGPGPFAEMGAMRIPAGHRL streptomyces sp. TMHYIAELGLQNQVREFRTLFSDDAAYLPSSAGYLRVREAHD TP-A0274 TLVDEFATGLPSAHYRQDTLLFGAWLDASIRAIAPRQFYDGL SEQ ID NO: 148 HNDIGVELLNLVDDIDLTPYRCGTARNRIDLHALFADHPRVR ASCPPRLERFLDDVLDETSSSIVRLKDGMDELPRRLASRIRGKI SLGQEVTGIDVHDDTVTLTVRQGLRTVTRTCDYVVCTIPFTVL RTLRLTGFDQDKLDIVHETKYWPATKIAFHCREPFWEKDGIS GGASFTGGHVRQTYYPPAEGDPALGAVLLASYTIGPDAEALA RMDEAERDALVAKELSVMHPELRRPGMVLAVAGRDWGARR WSRGAATVRWGQEAALREAERRECARPQKGLFFAGEHCSSK PAWIEGAIESAIDAAHEIEWYEPRASRVFAASRLSRSDRSA ipdC: Indole-3- MRTPYCVADYLLDRLTDCGADHLFGVPGDYNLQFLDHVIDS pyruvate PDICWVGCANELNASYAADGYARCKGFAALLTTFGVGELSA decarboxylase from MNGIAGSYAEHVPVLHIVGAPGTAAQQRGELLHHTLGDGEFR Enterobacter cloacae HFYHMSEPITVAQAVLTEQNACYEIDRVLTTMLRERRPGYLM SEQ ID NO: 149 LPADVAKKAATPPVNALTHKQAHADSACLKAFRDAAENKLA MSKRTALLADFLVLRHGLKHALQKWVKEVPMAHATMLMG KGIFDERQAGFYGTYSGSASTGAVKEAIEGADTVLCVGTRFT DTLTAGFTHQLTPAQTIEVQPHAARVGDVWFTGIPMNQAIET LVELCKQHVHAGLMSSSSGAIPFPQPDGSLTQENFWRTLQTFI RPGDIILADQGTSAFGAIDLRLPADVNFIVQPLWGSIGYTLAA AFGAQTACPNRRVIVLTGDGAAQLTIQELGSMLRDKQHPIILV LNNEGYTVERAIHGAEQRYNDIALWNWTHIPQALSLDPQSEC WRVSEAEQLADVLEKVAHHERLSLIEVMLPKADIPPLLGALT KALEACNNA IAD1: Indole-3- MPTLNLDLPNGIKSTIQADLFINNKFVPALDGKTFATINPSTGK acetaldehyde EIGQVAEASAKDVDLAVKAAREAFETTWGENTPGDARGRLLI dehydrogenase from KLAELVEANIDELAAIESLDNGKAFSIAKSFDVAAVAANLRY Ustilago maydis YGGWADKNHGKVMEVDTKRLNYTRHEPIGVCGQIIPWNFPL SEQ ID NO: 150 LMFAWKLGPALATGNTIVLKTAEQTPLSAIKMCELIVEAGFPP GVVNVISGFGPVAGAAISQHMDIDKIAFTGSTLVGRNIMKAA ASTNLKKVTLELGGKSPNIIFKDADLDQAVRWSAFGIMFNHG QCCCAGSRVYVEESIYDAFMEKMTAHCKALQVGDPFSANTF QGPQVSQLQYDRIMEYIESGKKDANLALGGVRKGNEGYFIEP TIFTDVPHDAKIAKEEIFGPVVVVSKFKDEKDLIRIANDSIYGL AAAVFSRDISRAIETAHKLKAGTVWVNCYNQLIPQVPFGGYK ASGIGRELGEYALSNYTNIKAVHVNLSQPAPI YUC2: indole-3- MEFVTETLGKRIHDPYVEETRCLMIPGPIIVGSGPSGLATAACL pyruvate KSRDIPSLILERSTCIASLWQHKTYDRLRLHLPKDFCELPLMPF monoxygenase from PSSYPTYPTKQQFVQYLESYAEHFDLKPVFNQTVEEAKFDRR Arabidopsis thaliana CGLWRVRTTGGKKDETMEYVSRWLVVATGENAEEVMPEID SEQ ID NO: 151 GIPDFGGPILHTSSYKSGEIFSEKKILVVGCGNSGMEVCLDLCN FNALPSLVVRDSVHVLPQEMLGISTFGISTSLLKWFPVHVVDR FLLRMSRLVLGDTDRLGLVRPKLGPLERKIKCGKTPVLDVGT LAKIRSGHIKVYPELKRVMHYSAEFVDGRVDNFDAIILATGY KSNVPMWLKGVNMFSEKDGFPHKPFPNGWKGESGLYAVGF TKLGLLGAAIDAKKIAEDIEVQRHFLPLARPQHC IaaM: Tryptophan 2- MYDHFNSPSIDILYDYGPFLKKCEMTGGIGSYSAGTPTPRVAI monooxygenase from VGAGISGLVAATELLRAGVKDVVLYESRDRIGGRVWSQVFD Pseudomonas QTRPRYIAEMGAMRFPPSATGLFHYLKKFGISTSTTFPDPGVV savastanoi DTELHYRGKRYHWPAGKKPPELFRRVYEGWQSLLSEGYLLE SEQ ID NO: 152 GGSLVAPLDITAMLKSGRLEEAAIAWQGWLNVFRDCSFYNAI VCIFTGRHPPGGDRWARPEDFELFGSLGIGSGGFLPVFQAGFT EILRMVINGYQSDQRLIPDGISSLAARLADQSFDGKALRDRVC FSRVGRISREAEKIIIQTEAGEQRVFDRVIVTSSNRAMQMIHCL TDSESFLSRDVARAVRETHLTGSSKLFILTRTKFWIKNKLPTTI QSDGLVRGVYCLDYQPDEPEGHGVVLLSYTWEDDAQKMLA MPDKKTRCQVLVDDLAAIHPTFASYLLPVDGDYERYVLHHD WLTDPHSAGAFKLNYPGEDVYSQRLFFQPMTANSPNKDTGL YLAGCSCSFAGGWIEGAVQTALNSACAVLRSTGGQLSKGNPL DCINASYRY iaaH: MHEIITLESLCQALADGEIAAAELRERALDTEARLARLNCFIRE Indoleacetamide GDAVSQFGEADHAMKGTPLWGMPVSFKDNICVRGLPLTAGT hydrolase from RGMSGFVSDQDAAIVSQLRALGAVVAGKNNMHELSFGVTSI Pseudomonas NPHWGTVGNPVAPGYCAGGSSGGSAAAVASGIVPLSVGTDT savastanoi GGSIRIPAAFCGITGFRPTTGRWSTAGIIPVSHTKDCVGLLTRT SEQ ID NO: 153 AGDAGFLYGLLSGKQQSFPLSRTAPCRIGLPVSMWSDLDGEV ERACVNALSLLRKTGFEFIEIDDADIVELNQTLTFTVPLYEFFA DLAQSLLSLGWKHGIHHIFAQVDDANVKGIINHHLGEGAIKP AHYLSSLQNGELLKRKMDELFARHNIELLGYPTVPCRVPHLD HADRPEFFSQAIRNTDLASNAMLPSITIPVGPEGRLPVGLSFDA LRGRDALLLSRVSAIEQVLGFVRKVLPHTT TrpDH: Tryptophan MLLFETVREMGHEQVLFCHSKNPEIKAIIAIHDTTLGPAMGAT dehydrogenase from RILPYINEEAALKDALRLSRGMTYKAACANIPAGGGKAVIIAN Nostoc punctiforme PENKTDDLLRAYGRFVDSLNGRFITGQDVNITPDDVRTISQET NIES-2108 KYVVGVSEKSGGPAPITSLGVFLGIKAAVESRWQSKRLDGMK SEQ ID NO: 154 VAVQGLGNVGKNLCRHLHEHDVQLFVSDVDPIKAEEVKRLF GATVVEPTEIYSLDVDIFAPCALGGILNSHTIPFLQASIIAGAAN NQLENEQLHSQMLAKKGILYSPDYVINAGGLINVYNEMIGYD EEKAFKQVHNIYDTLLAIFEIAKEQGVTTNDAARRLAEDRINN SKRSKSKAIAA CYP79B2: MNTFTSNSSDLTTTATETSSFSTLYLLSTLQAFVAITLVMLLKK tryptophan N- LMTDPNKKKPYLPPGPTGWPIIGMIPTMLKSRPVFRWLHSIMK monooxygenase from QLNTEIACVKLGNTHVITVTCPKIAREILKQQDALFASRPLTY Arabidopsis thaliana AQKILSNGYKTCVITPFGDQFKKMRKVVMTELVCPARHRWL SEQ ID NO: 155 HQKRSEENDHLTAWVYNMVKNSGSVDFRFMTRHYCGNAIK KLMFGTRTFSKNTAPDGGPTVEDVEHMEAMFEALGFTFAFCI SDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDERIKMWR EGKRTQIEDFLDIFISIKDEQGNPLLTADEIKPTIKELVMAAPDN PSNAVEWAMAEMVNKPEILRKAMEEIDRVVGKERLVQESDIP KLNYVKAILREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGS QVLLSRYGLGRNPKVWADPLCFKPERHLNECSEVTLTENDLR FISFSTGKRGCAAPALGTALTTMMLARLLQGFTWKLPENETR VELMESSHDMFLAKPLVMVGDLRLPEHLYPTVK CYP79B3: MDTLASNSSDLTTKSSLGMSSFTNMYLLTTLQALAALCFLMI tryptophan N- LNKIKSSSRNKKLHPLPPGPTGFPIVGMIPAMLKNRPVFRWLH monooxygenase from SLMKELNTEIACVRLGNTHVIPVTCPKIAREIFKQQDALFASRP Arabidopsis thaliana LTYAQKILSNGYKTCVITPFGEQFKKMRKVIMTEIVCPARHR SEQ ID NO: 156 WLHDNRAEETDHLTAWLYNMVKNSEPVDLRFVTRHYCGNA IKRLMFGTRTFSEKTEADGGPTLEDIEHMDAMFEGLGFTFAFC ISDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDERIKMWR EGKRTQIEDFLDIFISIKDEAGQPLLTADEIKPTIKELVMAAPDN PSNAVEWAIAEMINKPEILHKAMEEIDRVVGKERFVQESDIPK LNYVKAIIREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGSQV LLSRYGLGRNPKVWSDPLSFKPERHLNECSEVTLTENDLRFIS FSTGKRGCAAPALGTAITTMMLARLLQGFKWKLAGSETRVE LMESSHDMFLSKPLVLVGELRLSEDLYPMVK CYP71A13 : MSNIQEMEMILSISLCLTTLITLLLLRRFLKRTATKVNLPPSPW indoleacetaldoxime RLPVIGNLHQLSLHPHRSLRSLSLRYGPLMLLHFGRVPILVVSS dehydratase from GEAAQEVLKTHDHKFANRPRSKAVHGLMNGGRDVVFAPYG Arabidopis thaliana EYWRQMKSVCILNLLTNKMVESFEKVREDEVNAMIEKLEKA SEQ ID NO: 157 SSSSSSENLSELFITLPSDVTSRVALGRKHSEDETARDLKKRVR QIMELLGEFPIGEYVPILAWIDGIRGFNNKIKEVSRGFSDLMDK VVQEHLEASNDKADFVDILLSIEKDKNSGFQVQRNDIKFMILD MFIGGTSTTSTLLEWTMTELIRSPKSMKKLQDEIRSTIRPHGSY IKEKEVENMKYLKAVIKEVLRLHPSLPMILPRLLSEDVKVKGY NIAAGTEVIINAWAIQRDTAIWGPDAEEFKPERHLDSGLDYHG KNLNYIPFGSGRRICPGINLALGLAEVTVANLVGRFDWRVEA GPNGDQPDLTEAIGIDVCRKFPLIAFPSSVV PEN2: myrosinase MAHLQRTFPTEMSKGRASFPKGFLFGTASSSYQYEGAVNEGA from Arabidopsis RGQSVWDHFSNRFPHRISDSSDGNVAVDFYHRYKEDIKRMK thaliana DINMDSFRLSIAWPRVLPYGKRDRGVSEEGIKFYNDVIDELLA SEQ ID NO: 158 NEITPLVTIFHWDIPQDLEDEYGGFLSEQIIDDFRDYASLCFERF GDRVSLWCTMNEPWVYSVAGYDTGRKAPGRCSKYVNGASV AGMSGYEAYIVSHNMLLAHAEAVEVFRKCDHIKNGQIGIAHN PLWYEPYDPSDPDDVEGCNRAMDFMLGWHQHPTACGDYPE TMKKSVGDRLPSFTPEQSKKLIGSCDYVGINYYSSLFVKSIKH VDPTQPTWRTDQGVDWMKTNIDGKQIAKQGGSEWSFTYPTG LRNILKYVKKTYGNPPILITENGYGEVAEQSQSLYMYNPSIDT ERLEYIEGHIHAIHQAIHEDGVRVEGYYVWSLLDNFEWNSGY GVRYGLYYIDYKDGLRRYPKMSALWLKEFLRFDQEDDSSTS KKEEKKESYGKQLLHSVQDSQFVHSIKDSGALPAVLGSLFVV SATVGTSLFFKGANN Nit1: Nitrilase from MSSTKDMSTVQNATPFNGVAPSTTVRVTIVQSSTVYNDTPATI Arabidopsis thaliana DKAEKYIVEAASKGAELVLFPEGFIGGYPRGFRFGLAVGVHN SEQ ID NO: 159 EEGRDEFRKYHASAIHVPGPEVARLADVARKNHVYLVMGAI EKEGYTLYCTVLFFSPQGQFLGKHRKLMPTSLERCIWGQGDG STIPVYDTPIGKLGAAICWENRMPLYRTALYAKGIELYCAPTA DGSKEWQSSMLHIAIEGGCFVLSACQFCQRKHFPDHPDYLFT DWYDDKEHDSIVSQGGSVIISPLGQVLAGPNFESEGLVTADID LGDIARAKLYFDSVGHYSRPDVLHLTVNEHPRKSVTFVTKVE KAEDDSNK IDO1: indoleamine MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFI 2,3-dioxygenase from AKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLG homo sapiens CITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVY SEQ ID NO: 160 ADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVS LLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKA LQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGF WEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQ DMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDA CVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAK GTGGTDLMNFLKTVRSTTEKSLLKEG TDO2: tryptophan MSGCPFLGNNFGYTFKKLPVEGSEEDKSQTGVNRASKGGLIY 2,3-dioxygenase from GNYLHLEKVLNAQELQSETKGNKIHDEHLFIITHQAYELWFK homo sapiens QILWELDSVREIFQNGHVRDERNMLKVVSRMHRVSVILKLLV SEQ ID NO: 161 QQFSILETMTALDFNDFREYLSPASGFQSLQFRLLENKIGVLQ NMRVPYNRRHYRDNFKGEENELLLKSEQEKTLLELVEAWLE RTPGLEPHGFNFWGKLEKNITRGLEEEFIRIQAKEESEEKEEQV AEFQKQKEVLLSLFDEKRHEHLLSKGERRLSYRALQGALMIY FYREEPRFQVPFQLLTSLMDIDSLMTKWRYNHVCMVHRMLG SKAGTGGSSGYHYLRSTVSDRYKVFVDLFNLSTYLIPRHWIPK MNPTIHKFLYTAEYCDSSYFSSDESD BNA2: indoleamine MNNTSITGPQVLHRTKMRPLPVLEKYCISPHHGFLDDRLPLTR 2,3-dioxygenase from LSSKKYMKWEEIVADLPSLLQEDNKVRSVIDGLDVLDLDETIL S. cerevisiae GDVRELRRAYSILGFMAHAYIWASGTPRDVLPECIARPLLETA SEQ ID NO: 162 HILGVPPLATYSSLVLWNFKVTDECKKTETGCLDLENITTINTF TGTVDESWFYLVSVRFEKIGSACLNHGLQILRAIRSGDKGDA NVIDGLEGLAATIERLSKALMEMELKCEPNVFYFKIRPFLAGW TNMSHMGLPQGVRYGAEGQYRIFSGGSNAQSSLIQTLDILLG VKHTANAAHSSQGDSKINYLDEMKKYMPREHREFLYHLESV CNIREYVSRNASNRALQEAYGRCISMLKIFRDNHIQIVTKYIIL PSNSKQHGSNKPNVLSPIEPNTKASGCLGHKVASSKTIGTGGT RLMPFLKQCRDETVATADIKNEDKN Afmid: Kynurenine MAFPSLSAGQNPWRNLSSEELEKQYSPSRWVIHTKPEEVVGN formamidase from FVQIGSQATQKARATRRNQLDVPYGDGEGEKLDIYFPDEDSK mouse AFPLFLFLHGGYWQSGSKDDSAFMVNPLTAQGIVVVIVAYDI SEQ ID NO: 163 APKGTLDQMVDQVTRSVVFLQRRYPSNEGIYLCGHSAGAHL AAMVLLARWTKHGVTPNLQGFLLVSGIYDLEPLIATSQNDPL RMTLEDAQRNSPQRHLDVVPAQPVAPACPVLVLVGQHDSPE FHRQSKEFYETLLRVGWKASFQQLRGVDHFDIIENLTREDDV LTQIILKTVFQKL BNA3: kynurenine-- MKQRFIRQFTNLMSTSRPKVVANKYFTSNTAKDVWSLTNEA oxoglutarate AAKAANNSKNQGRELINLGQGFFSYSPPQFAIKEAQKALDIPM transaminase from S. VNQYSPTRGRPSLINSLIKLYSPIYNTELKAENVTVTTGANEGI cerevisae LSCLMGLLNAGDEVIVFEPFFDQYIPNIELCGGKVVYVPINPPK SEQ ID NO: 164 ELDQRNTRGEEWTIDFEQFEKAITSKTKAVIINTPHNPIGKVFT REELTTLGNICVKHNVVIISDEVYEHLYFTDSFTRIATLSPEIGQ LTLTVGSAGKSFAATGWRIGWVLSLNAELLSYAAKAHTRICF ASPSPLQEACANSINDALKIGYFEKMRQEYINKFKIFTSIFDEL GLPYTAPEGTYFVLVDFSKVKIPEDYPYPEEILNKGKDFRISH WLINELGVVAIPPTEFYIKEHEKAAENLLRFAVCKDDAYLEN AVERLKLLKDYL GOT2: Aspartate MALLHSGRVLPGIAAAFHPGLAAAASARASSWWTHVEMGPP aminotransferase, DPILGVTEAFKRDTNSKKMNLGVGAYRDDNGKPYVLPSVRK mitochondrial from AEAQIAAKNLDKEYLPIGGLAEFCKASAELALGENSEVLKSG homo sapiens RFVTVQTISGTGALRIGASFLQRFFKFSRDVFLPKPTWGNHTPI SEQ ID NO: 165 FRDAGMQLQGYRYYDPKTCGFDFTGAVEDISKIPEQSVLLLH ACAHNPTGVDPRPEQWKEIATVVKKRNLFAFFDMAYQGFAS GDGDKDAWAVRHFIEQGINVCLCQSYAKNMGLYGERVGAFT MVCKDADEAKRVESQLKILIRPMYSNPPLNGARIAAAILNTPD LRKQWLQEVKVMADRIIGMRTQLVSNLKKEGSTHNWQHITD QIGMFCFTGLKPEQVERLIKEFSIYMTKDGRISVAGVTSSNVG YLAHAIHQVTK AADAT: MNYARFITAASAARNPSPIRTMTDILSRGPKSMISLAGGLPNP Kynurenine/alpha- NMFPFKTAVITVENGKTIQFGEEMMKRALQYSPSAGIPELLSW aminoadipate LKQLQIKLHNPPTIHYPPSQGQMDLCVTSGSQQGLCKVFEMII aminotransferase, NPGDNVLLDEPAYSGTLQSLHPLGCNIINVASDESGIVPDSLR mitochondrial DILSRWKPEDAKNPQKNTPKFLYTVPNGNNPTGNSLTSERKK SEQ ID NO: 166 EIYELARKYDFLIIEDDPYYFLQFNKFRVPTFLSMDVDGRVIRA DSFSKITSSGLRIGFLTGPKPLIERVILHIQVSTLHPSTFNQLMIS QLLHEWGEEGFMAHVDRVIDFYSNQKDAILAAADKWLTGLA EWHVPAAGMFLWIKVKGINDVKELIEEKAVKMGVLMLPGN AFYVDSSAPSPYLRASFSSASPEQMDVAFQVLAQLIKESL CCLB1: Kynurenine- MAKQLQARRLDGIDYNPWVEFVKLASEHDVVNLGQGFPDFP -oxoglutarate PPDFAVEAFQHAVSGDFMLNQYTKTFGYPPLTKILASFFGELL transaminase 1 from GQEIDPLRNVLVTVGGYGALFTAFQALVDEGDEVIIIEPFFDC homo sapiens YEPMTMMAGGRPVFVSLKPGPIQNGELGSSSNWQLDPMELA SEQ ID NO: 167 GKFTSRTKALVLNTPNNPLGKVFSREELELVASLCQQHDVVCI TDEVYQWMVYDGHQHISIASLPGMWERTLTIGSAGKTFSATG WKVGWVLGPDHIMKHLRTVHQNSVFHCPTQSQAAVAESFER EQLLFRQPSSYFVQFPQAMQRCRDHMIRSLQSVGLKPIIPQGS YFLITDISDFKRKMPDLPGAVDEPYDRRFVKWMIKNKGLVAI PVSIFYSVPHQKHFDHYIRFCFVKDEATLQAMDEKLRKWKVE L CCLB2: kynurenine-- MFLAQRSLCSLSGRAKFLKTISSSKILGFSTSAKMSLKFTNAKR oxoglutarate IEGLDSNVWIEFTKLAADPSVVNLGQGFPDISPPTYVKEELSKI transaminase 3 from AAIDSLNQYTRGFGHPSLVKALSYLYEKLYQKQIDSNKEILVT homo sapiens VGAYGSLFNTIQALIDEGDEVILIVPFYDCYEPMVRMAGATPV SEQ ID NO: 168 FIPLRSKPVYGKRWSSSDWTLDPQELESKFNSKTKAIILNTPHN PLGKVYNREELQVIADLCIKYDTLCISDEVYEWLVYSGNKHL KIATFPGMWERTITIGSAGKTFSVTGWKLGWSIGPNHLIKHLQ TVQQNTIYTCATPLQEALAQAFWIDIKRMDDPECYFNSLPKEL EVKRDRMVRLLESVGLKPIVPDGGYFIIADVSLLDPDLSDMK NNEPYDYKFVKWMTKHKKLSAIPVSAFCNSETKSQFEKFVRF CFIKKDSTLDAAEEIIKAWSVQKS TnaA: tryptophanase MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE from E. coli DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE SEQ ID NO: 140 SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAM YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFP AQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRL TIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRH FTAKLKEV Trp MTATTISIETVPQAPAAGTKTNGTSGKYNPRTYLSDRAKVTEI aminotransferase DGSDAGRPNPDTFPFNSITLNLKPPLGLPESSNNMPVSITIEDPD (EC 2.6.1.27); LATALQYAPSAGIPKLREWLADLQAHVHERPRGDYAISVGSG tryptophan SQDLMFKGFQAVLNPGDPVLLETPMYSGVLPALRILKADYAE aminotransferase VDVDDQGLSAKNLEKVLSEWPADKKRPRVLYTSPIGSNPSGC [Cryptococcus SASKERKLEVLKVCKKYDVLIFEDDPYYYLAQELIPSYFALEK deuterogattii R265] QVYPEGGHVVRFDSFSKLLSAGMRLGFATGPKEILHAIDVSTA SEQ ID NO: 169 GANLHTSAVSQGVALRLMQYWGIEGFLAHGRAVAKLYTERR AQFEATAHKYLDGLATWVSPVAGMFLWIDLRPAGIEDSYELI RHEALAKGVLGVPGMAFYPTGRKSSHVRVSFSIVDLEDESDL GFQRLAEAIKDKRKALGLA Tryptophan MSQVIKKKRNTFMIGTEYILNSTQLEEAIKSFVHDFCAEKHEIH Decarboxylase (EC DQPVVVEAKEHQEDKIKQIKIPEKGRPVNEVVSEMMNEVYRY 4.1.1.28) Chain A, RGDANHPRFFSFVPGPASSVSWLGDIMTSAYNIHAGGSKLAP Ruminococcus MVNCIEQEVLKWLAKQVGFTENPGGVFVSGGSMANITALTA Gnavus ARDNKLTDINLHLGTAYISDQTHSSVAKGLRIIGITDSRIRRIPT SEQ ID NO: 170 NSHFQMDTTKLEEAIETDKKSGYIPFVVIGTAGTTNTGSIDPLT EISALCKKHDMWFHIDGAYGASVLLSPKYKSLLTGTGLADSIS WDAHKWLFQTYGCAMVLVKDIRNLFHSFHVNPEYLKDLEN DIDNVNTWDIGMELTRPARGLKLWLTLQVLGSDLIGSAIEHG FQLAVWAEEALNPKKDWEIVSPAQMAMINFRYAPKDLTKEE QDILNEKISHRILESGYAAIFTTVLNGKTVLRICAIHPEATQED MQHTIDLLDQYGREIYTEMKKa

TABLE 16B Tryptophan Pathway Catabolic Enzymes Description Sequence Trp ATGACGGCAACTACAATTTCTATTGAGACCGTACCTC aminotransferase AGGCCCCGGCGGCGGGGACCAAAACTAATGGGACTT (EC 2.6.1.27); CAGGAAAATACAACCCCCGCACTTACCTGTCCGACC tryptophan GCGCCAAAGTCACTGAGATTGATGGATCTGACGCCG aminotransferase GTCGCCCCAATCCCGATACTTTCCCATTTAACTCGAT [Cryptococcus TACCTTAAATTTGAAACCACCTTTAGGCTTGCCCGAG deuterogattii R265], AGTTCAAATAACATGCCGGTCTCTATCACGATTGAA codon optimized for GACCCCGATTTAGCGACGGCCTTACAATATGCACCT expression in E. coli AGCGCCGGTATTCCTAAGCTGCGCGAATGGCTGGCT SEQ ID NO: 171 GACTTACAAGCTCACGTTCATGAGCGCCCCCGTGGC GATTATGCCATCTCGGTCGGGTCGGGGTCACAGGAT TTGATGTTTAAGGGCTTCCAAGCTGTCTTGAATCCAG GTGATCCAGTCCTTCTGGAAACCCCAATGTATTCAGG TGTTCTGCCAGCGCTGCGCATTCTGAAGGCGGATTAT GCAGAAGTTGATGTAGACGACCAGGGGTTATCTGCT AAAAACCTTGAAAAAGTTTTATCAGAGTGGCCCGCA GATAAGAAGCGTCCTCGTGTCCTGTATACGTCGCCA ATCGGCTCCAATCCTTCCGGATGTTCAGCATCCAAGG AACGCAAGTTAGAGGTACTGAAAGTCTGTAAGAAGT ACGATGTGCTGATCTTCGAAGACGATCCGTATTATTA CCTTGCTCAAGAGCTTATTCCATCCTATTTTGCGTTG GAAAAACAAGTTTATCCGGAGGGTGGGCACGTTGTA CGCTTTGACTCATTTAGTAAATTGCTTTCTGCTGGGA TGCGCTTGGGATTTGCTACAGGGCCGAAGGAAATTC TTCATGCGATTGACGTCAGTACAGCAGGCGCAAATT TACATACTTCAGCGGTCTCTCAAGGTGTCGCTCTTCG CCTGATGCAGTATTGGGGGATCGAGGGATTCCTTGC ACATGGCCGCGCGGTGGCCAAACTTTACACGGAGCG CCGCGCTCAGTTCGAGGCAACCGCACATAAGTACCT GGACGGGCTGGCCACTTGGGTATCTCCCGTAGCGGG AATGTTTTTATGGATCGATCTTCGTCCAGCAGGAATC GAAGATTCTTACGAATTAATTCGCCATGAAGCATTA GCCAAAGGCGTTTTAGGCGTTCCAGGGATGGCGTTTT ATCCGACAGGCCGTAAGTCTTCCCATGTTCGTGTCAG TTTCAGTATCGTCGACCTGGAAGACGAATCTGACCTT GGTTTTCAACGCCTGGCTGAAGCTATTAAGGATAAA CGCAAGGCTTTAGGGCTGGCT Tryptophan ATGAGTCAAGTGATTAAGAAGAAACGTAACACCTTT Decarboxylase (EC ATGATCGGAACGGAGTACATTCTTAACAGTACACAA 4.1.1.28) Chain A, TTGGAGGAAGCGATTAAATCATTCGTACATGATTTCT Ruminococcus GCGCAGAGAAGCATGAGATCCATGATCAACCTGTGG Gnavus Tryptophan TAGTAGAAGCTAAAGAACATCAGGAGGACAAAATC Decarboxylase Rum AAACAAATCAAAATCCCGGAAAAGGGACGTCCTGTA gna_01526 (alpha- AATGAAGTCGTTTCTGAGATGATGAATGAAGTGTAT fmt); codon CGCTACCGCGGAGACGCCAACCATCCTCGCTTTTTTT optimized for the CTTTTGTGCCCGGACCTGCAAGGAGTGTGTCGTGGTT expression in E. coli GGGGGATATTATGACGTCCGCCTACAATATrCATGCT GGAGGCTCAAAGCTGGCACCGATGGTTAACTGCATT GAGCAGGAAGTTCTGAAGTGGTTAGCAAAGGAAGTG GGGTTCACAGAAAATCCAGGTGGCGTATTTGTGTCG GGCGGTTCAATGGCGAATATTACGGCACTTACTGCG GCTCGTGACAATAAACTGACCGACATTAACCTTCATT TGGGAACTGCTTATATTAGTGACCAGACTCATAGTTC AGTTGCGAAAGGATTACGCATTATTGGAATCACTGA CAGTCGCATCCGTCGCATTCCCACTAACTCCCACTTC CAGATGGATACCACCAAGCTGGAGGAAGCCATCGAG ACCGACAAGAAGTCTGGCTACATTCCGTTCGTCGTTA TCGGAACAGCAGGTACCACCAACACTGGTTCGATTG ACCCCCTGACAGAAATCTCTGCGTTATGTAAGAAGC ATGACATGTGGTTTCATATCGACGGAGCGTATGGAG CTAGTGTTCTGCTGTCACCTAAGTACAAGAGCCTTCT TACCGGAACCGGCTTGGCTGACAGTATTTCGTGGGA TGCTCATAAATGGTTGTTCCAAACGTACGGCTGTGCA ATGGTACTTGTCAAAGATATCCGTAATTTATTCCACT CTTTTCATGTGAATCCCGAGTATCTTAAGGATCTGGA AAACGACATCGATAACGTTAATACATGGGACATCGG CATGGAGCTGACGCGCCCTGCACGCGGTCTTAAATT GTGGCTTACTTTACAGGTCCTTGGATCTGACTTGATT GGGAGTGCCATTGAACACGGTTTCCAGCTGGCAGTT TGGGCTGAGGAACCATTGAATCCAAAGAAAGACTGG GAGATCGTTTCTCCAGCTCAGATGGCTATGATTAATT TCCGTTATGCCCCTAAGGATTTAACCAAAGAGGAAC AGGATATTCTGAATGAAAAGATCTCCCACCGCATTTT AGAGAGCGGATACGCTGCAATTTTCACTACTGTATTA AACGGCAAGACCGTTTTACGCATCTGTGCAATTCACC CGGAGGCAACTCAAGAGGATATGCAACACAATCG ACTTATTAGACCAATACGGTCGTGAAATCTATACCG AGATGAAGAAAGCG

In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 16B or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as listed in Table 16A or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 16B or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide the polypeptide sequences listed in Table 16A or a functional fragment thereof.

In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 141. In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 141. In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 141. Accordingly, in one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 141. In yet another embodiment the Tryptophan Decarboxylase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 141.

In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 149. In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 149. In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 149. Accordingly, in one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 149. In yet another embodiment the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 149.

In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 150. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 150. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 150. Accordingly, in one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 150. In yet another embodiment the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 150.

In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 154. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 154. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 154. Accordingly, in one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 154. In yet another embodiment the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 154.

In one embodiment, genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 80% identity with the entire sequence of one or more sequence(s) of Table 16A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 85% identity with the entire sequence of one or more sequence(s) of Table 16A. In one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 90% identity with the entire sequence of one or more sequence(s) of Table 16A. In one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 95% identity with the entire sequence of one or more sequence(s) of Table 16A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 96%, 97%, 98%, or 99%identity with the entire sequence of one or more sequence(s) of Table 16A. Accordingly, in one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more sequence(s) of Table 16A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which comprises the entire sequence of one or more sequence(s) of Table 16A.

In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In som embodiments the bacteria further produce tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptamine exporter. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

Table 17 depicts non-limiting examples of contemplated polypeptide sequences, which are encoded by indole-3-propionate producing bacteria.

TABLE 17 Non-limiting Examples of Sequences for indole-3-propionate Production Description Sequence FldA: indole-3- MENNTNMFSGVKVIELANFIAAPAAGRFFADGGAEVIKIESPA propionyl- GDPLRYTAPSEGRPLSQEENTTYDLENANKKAIVLNLKSEKGK CoA: indole-3- KILHEMLAEADILLTNWRTKALVKQGLDYETLKEKYPKLVFA lactate CoA QITGYGEKGPDKDLPGFDYTAFFARGGVSGTLYEKGTVPPNV transferase from VPGLGDHQAGMFLAAGMAGALYKAKTTGQGDKVTVSLMHS Clostridium AMYGLGIMIQAAQYKDHGLVYPINRNETPNPFIVSYKSKDDYF sporogenes VQVCMPPYDVFYDRFMTALGREDLVGDERYNKIENLKDGRA SEQ ID NO: 173 KEVYSIIEQQMVTKTKDEWDKIFRDADIPFAIAQTWEDLLEDE QAWANDYLYKMKYPTGNERALVRLPVFFKEAGLPEYNQSPQI AENTVEVLKEMGYTEQEIEELEKDKDIMVRKEK FldB: subunit of MSDRNKEVKEKKAKHYLREITAKHYKEALEAKERGEKVGWC indole-3-lactate ASNFPQEIATTLGVKVVYPENHAAAVAARGNGQNMCEHAEA dehydratase from MGFSNDVCGYARVNLAVMDIGHSEDQPIPMPDFVLCCNNICN Clostridium QMIKWYEHIAKTLDIPMILIDIPYNTENTVSQDRIKYIRAQFDD sporogenes AIKQLEEITGKKWDENKFEEVMKISQESAKQWLRAASYAKYK SEQ ID NO: 174 PSPFSGFDLFNHMAVAVCARGTQEAADAFKMLADEYEENVKT GKSTYRGEEKQRILFEGIACWPYLRHKLTKLSEYGMNVTATV YAEAFGVIYENMDELMAAYNKVPNSISFENALKMRLNAVTST NTEGAVIHINRSCKLWSGFLYELARRLEKETGIPVVSFDGDQA DPRNFSEAQYDTRIQGLNEVMVAKKEAE FldC: subunit of MSNSDKFFNDFKDIVENPKKYIMKHMEQTGQKAIGCMPLYTP indole-3-lactate EELVLAAGMFPVGVWGSNTELSKAKTYFPAFICSILQTTLENA dehydratase from LNGEYDMLSGMMITNYCDSLKCMGQNFKLTVENIEFIPVTVPQ Clostridium NRKMEAGKEFLKSQYKMNIEQLEKISGNKITDESLEKAIEIYDE sporogenes HRKVMNDFSMLASKYPGIITPTKRNYVMKSAYYMDKKEHTE SEQ ID NO: 175 KVRQLMDEIKAIEPKPFEGKRVITTGIIADSEDLLKILEENNIAIV GDDIAHESRQYRTLTPEANTPMDRLAEQFANRECSTLYDPEKK RGQYIVEMAKERKADGIIFFMTKFCDPEEYDYPQMKKDFEEA GIPHVLIETDMQMKNYEQARTAIQAFSETL FldD: indole-3- MFFTEQHELIRKLARDFAEQEIEPIADEVDKTAEFPKEIVKKMA acrylyl-CoA QNGFFGIKMPKEYGGAGADNRAYVTIMEEISRASGVAGIYLSS reductase from PNSLLGTPFLLVGTDEQKEKYLKPMIRGEKTLAFALTEPGAGS Clostridium DAGALATTAREEGDYYILNGRKTFITGAPISDNIIVFAKTDMSK sporogenes GTKGITTFIVDSKQEGVSFGKPEDKMGMIGCPTSDIILENVKVH SEQ ID NO: 176 KSDILGEVNKGFITAMKTLSVGRIGVASQALGIAQAAVDEAVK YAKQRKQFNRPIAKFQAIQFKLANMETKLNAAKLLVYNAAYK MDCGEKADKEASMAKYFAAESAIQIVNDALQIHGGYGYIKDY KIERLYRDVRVIAIYEGTSEVQQMVIASNLLK FldH1: indole-3- MKILAYCVRPDEVDSFKKFSEKYGHTVDLIPDSFGPNVAHLAK lactate GYDGISILGNDTCNREALEKIKDCGIKYLATRTAGVNNIDFDA dehydrogenase AKEFGINVANVPAYSPNSVSEFTIGLALSLTRKIPFALKRVELN from Clostridium NFALGGLIGVELRNLTLGVIGTGRIGLKVIEGFSGFGMKKMIGY sporogenes DIFENEEAKKYIEYKSLDEVFKEADIITLHAPLTDDNYHMIGKE SEQ ID NO: 177 SIAKMKDGVFIINAARGALIDSEALIEGLKSGKIAGAALDSYEY EQGVFHNNKMNEIMQDDTLERLKSFPNVVITPHLGFYTDEAVS NMVEITLMNLQEFELKGTCKNQRVCK FldH2: indole-3- MKILMYSVREHEKPAIKKWLEANPGVQIDLCNNALSEDTVCK lactate AKEYDGIAIQQTNSIGGKAVYSTLKEYGIKQIASRTAGVDMIDL dehydrogenase KMASDSNILVTNVPAYSPNAIAELAVTHTMNLLRNIKTLNKRI from Clostridium AYGDYRWSADLIAREVRSVTVGVVGTGKIGRTSAKLFKGLGA sporogenes NVIGYDAYPDKKLEENNLLTYKESLEDLLREADVVTLHTPLLE SEQ ID NO: 178 STKYMINKNNLKYMKPDAFIVNTGRGGIINTEDLIEALEQNKIA GAALDTFENEGLFLNKVVDPTKLPDSQLDKLLKMDQVLITHH VGFFTTTAVQNIVDTSLDSVVEVLKTNNSVNKVN AcuI: acrylyl- MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYK CoA reductase DALAITGKAPVVRRFPMVPGIDFTGTVAQSSHADFKPGDRVIL from Rhodobacter NGWGVGEKHWGGLAERARVRGDWLVPLPAPLDLRQAAMIG sphaeroides TAGYTAMLCVLALERHGVVPGNGEIVVSGAAGGVGSVATTLL SEQ ID NO: 179 AAKGYEVAAVTGRASEAEYLRGLGAASVIDRNELTGKVRPLG QERWAGGIDVAGSTVLANMLSMMKYRGVVAACGLAAGMDL PASVAPFILRGMTLAGVDSVMCPKTDRLAAWARLASDLDPAK LEEMTTELPFSEVIETAPKFLDGTVRGRIVIPVTP

In one embodiment, the tryptophan pathway catabolic enzyme encoded by the genetically engineered bacteria has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 85% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 90% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 95% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. Accordingly, in one embodiment, the tryptophan pathway catabolic enzyme has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme comprises the sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In yet another embodiment the tryptophan pathway catabolic enzyme consists of the sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179.

In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of one or more indole pathway metabolites described herein from tryptophan or a tryptophan metabolite. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments, the genetically engineered bacteria additionally produce tryptophan and/or chorismate through any of the pathways described herein, e.g. FIG. 39, FIG. 45A and FIG. 45B. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose or tetracycline. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. In some embodiments, the tryptophan synthesis and/or tryptophan catabolism cassette(s) is under control of an inducible promoter. Exemplary inducible promoters which may control the expression of the al teast one sequence(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more exporters for exporting biological molecules or substrates, such any of the exporters described herein or otherwise known in the art, (6) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (7) combinations of one or more of such additional circuits.

Tryptophan Repressor (TrpR)

In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, e.g., sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Tryptophan and Tryptophan MetaboliteTransport

Metabolite transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance tryptophan or KP metabolite transport into the cell.

The inner membrane protein YddG of E. coli , encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbiol. Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

In some embodiments, the engineered microbe has a mechanism for importing (transporting) Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene.

In some embodiments, the genetically engineered bacteria comprise a transporter to facilitate uptake of tryptophan into the cell. Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli. In some embodiments, the genetically engineered bacteria comprise one or more copies of one or more of Mtr, TnaB, and AroP.

In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the transporter gene. In some embodiments, the genetically engineered bacteria of the invention also comprise a transporte gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of a transporter gene from a different bacterial species. In some embodiments, the native transporter gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise a transporter gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter.

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

In some embodiments, the native transporter gene is mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized transporter gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native transporter gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The transporter modifications described herein may be present on a plasmid or chromosome.

In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, is inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter.

Inhibitory and Targeting Molecules

In some embodiments, the genetically engineered bacteria of the invention are capable of producing a molecule that is capable of inhibiting a metabolic disease-promoting molecule. The genetically engineered bacteria may express any suitable inhibitory molecule, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA, that is capable of neutralizing one or more metabolic disease-promoting molecules, e.g., dipeptidyl peptidase-4 (DPP-4) or ghrelin receptor. The genetically engineered bacteria may inhibit one or more metabolic disease-promoting molecules.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in plants and animals. RNAi is activated when microRNA (miRNA), double-stranded RNA (dsRNA), or short hairpin RNA (shRNA) is processed into short interfering RNA (siRNA) duplexes (Keates et al., 2008). RNAi can be “activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver shRNA to target cells” such as mammalian cells (Keates et al., 2008). In some embodiments, the genetically engineered bacteria of the invention induce RNAi-mediated gene silencing of one or more metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.In some embodiments, the genetically engineered bacteria produce siRNA targeting DPP-4 in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Single-chain variable fragments (scFv) are “widely used antibody fragments . . . produced in prokaryotes” (Frenzel et al., 2013). scFv lacks the constant domain of a traditional antibody and expresses the antigen-binding domain as a single peptide. Bacteria such as Escherichia coli are capable of producing scFv that target a variety of molecules, e.g., TNF (Hristodorov et al., 2014). In some embodiments, the genetically engineered bacteria of the invention express a binding protein for neutralizing one or more metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the genetically engineered bacteria produce scFv targeting DPP-4 in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the genetically engineered bacteria produce both scFv and siRNA targeting one or more metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose (see, e.g., Xiao et al., 2014).

In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.

Generation of Bacterial Strains with Enhanced Ability to Transport Amino Acids

Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population.

This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite.

For example, if the biosynthetic pathway for producing an amino acid is disrupted a strain capable of high-affinity capture of said amino acid can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the amino acid at regular intervals. Over time, cells that are most competitive for the amino acid—at growth-limiting concentrations—will come to dominate the population. These strains will likely have mutations in their amino acid-transporters resulting in increased ability to import the essential and limiting amino acid.

Similarly, by using an auxotroph that cannot use an upstream metabolite to form an amino acid, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream metabolite. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways.

A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate.

Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth-limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth.

Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures.

Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations “screened” throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 1011.2 CCD1. This rate can be accelerated by the addition of chemical mutagens to the cultures—such as N-methyl-N-nitro-N-nitrosoguanidine (NTG)—which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted.

At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvoluted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. Ø. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

Similar methods can be used to generate E. Coli Nissle mutants that consume or import tryptophan and/or kynurenine.

Regulation of Payload Expression

In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes. In some embodiments, bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes. In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes.

Herein the term “payload” refers to one or more effector molecules described herein and/or one or more enzyme(s) or polypeptide(s0 needed for the production of such effector molecules. Non-limiting examples of payloads include butyrate, propionate, acetate, and butyrate and/or propionate and/or acetate producing enzymes,. Further examples include GLP-1, GLP-2, manganese transporters, GABA transporters, tryptophan and/pr tryptophan metabolite transporters, aromatic amino acid transporters, and polypeptides for metabolizing (catabolizing) GABA. Yet further examples include tryptophan and/or one or more of its metabolites, e.g., KP metabolites ann/or indole metabolites as described herein, and/or one or more enzyme(s) for the production of tryptophan and/or one or more of its metabolites, and/or one or more gut-barrier enhancing molecule(s) and/or antinflammatory molecules described herein. Yet other examples include bile salt hydrolases, bile salte transporters, and bile salt exporters described herein.

In some embodiments, the genetically engineered bacteria comprise multiple copies of the same payload gene(s). In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.

In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on chromosome and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.

In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is induced under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.

In some embodiments, the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manuafacturing conditions). In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manuafacturing conditions).

In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. . In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell.

FNR dependent Regulation

The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule, wherein the gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is controlled by exogenous environmental condition(s). In some embodiments, the inducible promoter is an oxygen level-dependent promoter and the metabolic and/or satiety effector and/or immune modulator molecule is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, in low oxygen conditions, the oxygen level-dependent promoter is activated by a corresponding oxygen level-sensing transcription factor, thereby driving production of the metabolic and/or satiety effector and/or immune modulator molecule

Bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An oxygen level-dependent promoter is a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression. In one embodiment, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter. In a more specific aspect, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut.

In certain embodiments, the bacterial cell comprises a gene encoding a payload expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. In E. coli , FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive. FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in Table 18 and Table 19 below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.

TABLE 18 FNR Promoter Sequences FNR Responsive Promoter Sequence SEQ ID NO: 180 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCA CTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTT CTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCA GACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCC TTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTT GCTGAATCGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA SEQ ID NO: 181 ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGG CTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAA TATTTCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCG ACTGTAAATCAGAAAGGAGAAAACACCT SEQ ID NO: 182 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCA CTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTT CTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCA GACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCC TTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTT GCTGAATCGTTAAGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAG AAGGAGATATACAT SEQ ID NO: 183 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATG GCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAA ATATTTCACTCGACAGGAGTATTTATATTGCGCCCGGATCC CTCTAGA AATAATTTTGTTTAACTTTAAGAAGGAGATATACAT SEQ ID NO: 184 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGG TTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTA AAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCT CTCTTGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGAT ATACAT

TABLE 19 FNR Promoter sequences FNR-responsive regulatory region 12345678901234567890123456789012345678901234567890 SEQ ID NO: 185 ATCCCCATCACTCTTGATGGAGATCAATTCCCCAAGCTGCTA GAGCGTTACCTTGCCCTTAAACATTAGCAATGTCGATTTATC AGAGGGCCGACAGGCTCCCACAGGAGAAAACCG SEQ ID NO: 186 CTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTAC CTTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCG ACAGGCTCCCACAGGAGAAAACCG nirB1 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGG SEQ ID NO: 187 GCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTA CGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTT GCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAA ATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAA AGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATC GTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA nirB2 CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGC SEQ ID NO: 188 TTACAGCAAACGGTCTGTACGCTGTCGTCTTTGTGATGTGCT TCCTGTTAGGTTTCGTCAGCCGTCACCGTCAGCATAACACCC TGACCTCTCATTAATTGCTCATGCCGGACGGCACTATCGTCG TCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCT ATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAA TATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCA GCAATATACCCATTAAGGAGTATATAAAGGTGAATTTGATTT ACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGT AATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaagaaggagatat acat nirB3 GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGG SEQ ID NO: 189 ACGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTA CGTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTT GCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAA ATTTATACAAATCAGCAATATACCCATTAAGGAGTATATAA AGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATC GTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA ydfZ ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGA SEQ ID NO: 190 CTTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATC AAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATTG CGCCCGTTACGTGGGCTTCGACTGTAAATCAGAAAGGAGAA AACACCT nirB + RBS GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGG SEQ ID NO: 191 GCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTA CGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTT GCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAA ATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAA AGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATC GTTAAGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAGAA GGAGATATACAT ydfZ + RBS CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCG SEQ ID NO: 192 ACTTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGA TCAAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATT GCGCCCGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAGA AGGAGATATACAT fnrS1 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT SEQ ID NO: 193 AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATAC AAAAACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTA CCCATTCAGGGCAATATCTCTCTTGGATCC CTCTAGAAATA ATTTTGTTTAACTTTAAGAAGGAGATATACAT fnrS2 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT SEQ ID NO: 194 AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATAC AAAAACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTA CCCATTCAGGGCAATATCTCTCTTGGATCCAAAGTGAACTCT AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT nirB + crp TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCA SEQ ID NO: 195 CCGTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCC GGACGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCG CTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTT TTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAG AAAATTTATACAAATCAGCAATATACCCATTAAGGAGTATA TAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGA ATCGTTAAGGTAGaaatgtgatctagttcacatttGCGGTAATAGAAAAG AAATCGAGGCAAAAatgtttgtttaactttaagaaggagatatacat fnrS + crp AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT SEQ ID NO: 196 AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATAC AAAAACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTA CCCATTCAGGGCAATATCTCTCaaatgtgatctagttcacattttttgtttaacttta agaaggagatatacat

FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload.

As used herein the term “payload” refers to one or more e.g. anti-inflammation and/or gut barrier function enhancer molecule(s), including but not limited to, butyrate, propionate, acetate, IL10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, SOD, GLP2, GLP1, and/or tryptophan and/or its metabolites. As used herein, the term “polypeptide of interest” or “polypeptides of interest”, “protein of interest”, “proteins of interest”, “payload”, “payloads” further includes any or a plurality of any of the short chain fatty acid producing enzymes, trypophan synthesis, tryptophan metabolite producing enzymes, or bile salt hydrolases and/or bile salt transporters or exporters, enzymes producing any gut barrier enhancer and/or anti-inflammatory metabolite, metabolite transporters or exporters, described herein. As used herein, the term “gene of interest” or “gene sequence of interest” includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more anti-inflammation and/or gut barrier function enhancer molecule(s) described herein.

Non-limiting FNR promoter sequences are provided in Table 6. Table 6 depicts the nucleic acid sequences of exemplary regulatory region sequences comprising a FNR-responsive promoter sequence. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, nirB1 promoter (SEQ ID NO: 187), nirB2 promoter (SEQ ID NO: 188), nirB3 promoter (SEQ ID NO: 189), ydfZ promoter (SEQ ID NO: 190), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 191), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 192), fnrS, an anaerobically induced small RNA gene (fnrS1 promoter SEQ ID NO: 193 or fnrS2 promoter SEQ ID NO: 194), nirB promoter fused to a crp binding site (SEQ ID NO: 195), and fnrS fused to a crp binding site (SEQ ID NO: 196).

In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a payload expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997). In these embodiments, expression of the payload gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut.

In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule expressed under the control of anaerobic regulation of arginine deiminiase and nitrate reduction transcriptional regulator (ANR). In P. aeruginosa, ANR is “required for the expression of physiological functions which are inducible under oxygen-limiting or anaerobic conditions” (Winteler et al., 1996; Sawers 1991). P. aeruginosa ANR is homologous with E. coli FNR, and “the consensus FNR site (TTGAT----ATCAA) was recognized efficiently by ANR and FNR” (Winteler et al., 1996). Like FNR, in the anaerobic state, ANR activates numerous genes responsible for adapting to anaerobic growth. In the aerobic state, ANR is inactive. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all have functional analogs of ANR (Zimmermann et al., 1991). Promoters that are regulated by ANR are known in the art, e.g., the promoter of the arcDABC operon (see, e.g., Hasegawa et al., 1998).

In other embodiments, the one or more gene sequence(s) for producing a payload are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g., CRP. CRP (cyclic AMP receptor protein or catabolite activator protein or CAP) plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al., 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Görke and Stülke, 2008). In some embodiments, the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, the one or more gene sequence(s) for a payload are controlled by a FNR promoter fused to a CRP binding site. In these embodiments, cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions. In the presence of glucose, cyclic AMP does not bind to CRP and transcription of the gene or gene cassette for producing an payload is repressed. In some embodiments, an oxygen level-dependent promoter (e.g., an FNR promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing an payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g., by adding glucose to growth media in vitro.

In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria. The heterologous oxygen-level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g., one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g., Isabella et al., 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.

In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., (2006). In some embodiments, both the oxygen level-sensing transcriptional regulator and corresponding promoter are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in low-oxygen conditions.

In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid.

In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload. In some embodiments, the transcriptional regulator and the payload are divergently transcribed from a promoter region.

RNS-Dependent Regulation

In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene encoding a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the payload is expressed under the control of an inflammatory-dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter.

As used herein, “reactive nitrogen species” and “RNS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO●), peroxynitrite or peroxynitrite anion (ONOO—), nitrogen dioxide (●NO2), dinitrogen trioxide (N2O3), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOCO2—) (unpaired electrons denoted by ●). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.

As used herein, “RNS-inducible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS- inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s), e.g., any of the payloads described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.

As used herein, “RNS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.

As used herein, “RNS-repressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS-repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.

As used herein, a “RNS-responsive regulatory region” refers to a RNS-inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 20.

TABLE 20 Examples of RNS-sensing transcription factors and RNS-responsive genes Examples of responsive genes, RNS-sensing Primarily capable promoters, and/or regulatory transcription factor: of sensing: regions: NsrR NO norB, aniA, nsrR, hmpA, ytfE, ygbA, hcp, hcr, nrfA, aox NorR NO norVW, norR DNR NO norCB, nir, nor, nos

1.

In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a payload, such as any of the payloads provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the payload gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload is decreased or eliminated.

In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.

In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR “is an NO-responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide” (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more payload gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the payload(s).

In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR (dissimilatory nitrate respiration regulator) “promotes the expression of the nir, the nor and the nos genes” in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more payloads. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.

In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule expressed under the control of the dissimilatory nitrate respiration regulator (DNR). DNR is a member of the FNR family (Arai et al., 1995) and is a transcriptional regulator that is required in conjunction with ANR for “anaerobic nitrate respiration of Pseudomonas aeruginosa” (Hasegawa et al., 1998). For certain genes, the FNR-binding motifs “are probably recognized only by DNR” (Hasegawa et al., 1998). Any suitable transcriptional regulator that is controlled by exogenous environmental conditions and corresponding regulatory region may be used. Non-limiting examples include ArcA/B, ResD/E, NreA/B/C, and AirSR, and others are known in the art.

In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is “an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism” (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a payload gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked a payload gene or genes and producing the encoding a payload(s).

In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae, wherein the Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g., encoding a payload. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments, include, but are not limited to, TetR, C1, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g., a payload gene or genes is expressed.

A RNS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011; Karlinsey et al., 2012).

In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type activity.

In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g., the NsrR gene, and a corresponding regulatory region, e.g., a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS-responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g., NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of RNS.

In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of one or more encoding a payload gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the payload(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

In some embodiments, the genetically engineered bacteria of the invention produce at least one payload in the presence of RNS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold as compared to unmodified bacteria of the same subtype under the same conditions. Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g., by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen).

In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of payload in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS.

ROS-Dependent Regulation

In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene for producing a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the payload is expressed under the control of an cellular damaged-dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.

As used herein, “reactive oxygen species” and “ROS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal-catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H2O2), organic peroxide (ROOH), hydroxyl ion (OH—), hydroxyl radical (●OH), superoxide or superoxide anion (●O2-), singlet oxygen (1O2), ozone (O3), carbonate radical, peroxide or peroxyl radical (●O2-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOC1), nitric oxide (NO●), and peroxynitrite or peroxynitrite anion (ONOO—) (unpaired electrons denoted by ●). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al., 2014).

As used herein, “ROS-inducible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g., a sequence or sequences encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OxyR, senses ROS and activates a corresponding ROS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.

As used herein, “ROS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS-derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.

As used herein, “ROS-repressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS-repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.

As used herein, a “ROS-responsive regulatory region” refers to a ROS-inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 21.

TABLE 21 Examples of ROS-sensing transcription factors and ROS-responsive genes ROS-sensing Examples of responsive genes, transcription Primarily capable promoters, and/or regulatory factor: of sensing: regions: OxyR H₂O₂ ahpC; ahpF; dps; dsbG; fhuF; flu; fur; gor; grxA; hemH; katG; oxyS; sufA; sufB; sufC; sufD; sufE; sufS; trxC; uxuA; yaaA; yaeH; yaiA; ybjM; ydcH; ydeN; ygaQ; yljA; ytfK PerR H₂O₂ katA; ahpCF; mrgA; zoaA; fur; hemAXCDBL; srfA OhrR Organic peroxides ohrA NaOCl SoxR •O₂ ⁻ soxS NO• (also capable of sensing H₂O₂) RosR H₂O₂ rbtT; tnp16a; rluC1; tnp5a; mscL; tnp2d; phoD; tnp15b; pstA; tnp5b; xylC; gabD1; rluC2; cgtS9; azlC; narKGHJI; rosR

2.

In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is a payload; when ROS is present, e.g., in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the payload, thereby producing the payload. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload is decreased or eliminated.

In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.

In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR “functions primarily as a global regulator of the peroxide stress response” and is capable of regulating dozens of genes, e.g., “genes involved in H2O2 detoxification (katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe-S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)” and “OxyS, a small regulatory RNA” (Dubbs et al., 2012). The genetically engineered bacteria may comprise any suitable ROS-responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a payload gene. In the presence of ROS, e.g., H2O2, an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked payload gene and producing the payload. In some embodiments, OxyR is encoded by an E. coli oxyR gene. In some embodiments, the oxyS regulatory region is an E. coli oxyS regulatory region. In some embodiments, the ROS-inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.

In alternate embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is “activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression” (Koo et al., 2003). “SoxR is known to respond primarily to superoxide and nitric oxide” (Koo et al., 2003), and is also capable of responding to H2O2. The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al., 2003). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., a payload. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked a payload gene and producing the a payload.

In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR “binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event,” but oxidized OhrR is “unable to bind its DNA target” (Duarte et al., 2010). OhrR is a “transcriptional repressor [that] . . . senses both organic peroxides and NaOC1” (Dubbs et al., 2012) and is “weakly activated by H2O2 but it shows much higher reactivity for organic hydroperoxides” (Duarte et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g., a payload gene. In the presence of ROS, e.g., NaOC1, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked payload gene and producing the a payload.

OhrR is a member of the MarR family of ROS-responsive regulators. “Most members of the MarR family are transcriptional repressors and often bind to the −10 or −35 region in the promoter causing a steric inhibition of RNA polymerase binding” (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al., 2012).

In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is “a MarR-type transcriptional regulator” that binds to an “18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA” and is “reversibly inhibited by the oxidant H2O2” (Bussmann et al., 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to “a putative polyisoprenoid-binding protein (cg1322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cg1426), two putative FMN reductases (cg1150 and cg1850), and four putative monooxygenases (cg0823, cg1848, cg2329, and cg3084)” (Bussmann et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g., a payload. In the presence of ROS, e.g., H2O2, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked payload gene and producing the payload.

In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR “when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)” (Marinho et al., 2014). PerR is a “global regulator that responds primarily to H202” (Dubbs et al., 2012) and “interacts with DNA at the per box, a specific palindromic consensus sequence (TTATAATNATTATAA) residing within and near the promoter sequences of PerR-controlled genes” (Marinho et al., 2014). PerR is capable of binding a regulatory region that “overlaps part of the promoter or is immediately downstream from it” (Dubbs et al., 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et al., 2012).

In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a payload. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, C1, and LexA. In some embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a payload. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a payload. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a payload, is expressed.

A ROS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although “OxyR is primarily thought of as a transcriptional activator under oxidizing conditions . . . OxyR can function as either a repressor or activator under both oxidizing and reducing conditions” (Dubbs et al., 2012), and OxyR “has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)” (Zheng et al., 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et al., 2001). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by RosR. In addition, “PerR-mediated positive regulation has also been observed . . . and appears to involve PerR binding to distant upstream sites” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by PerR.

One or more types of ROS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. For example, “OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively. One ROS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.

Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 22. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, or SEQ ID NO: 200, or a functional fragment thereof.

TABLE 22 Nucleotide sequences of exemplary OxyR-regulated regulatory regions Regulatory sequence Sequence katG TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTTAAACA (SEQ ID GAGCACAAAATGCTGCCTCGAAATGAGGGCGGGAAAATAAGGT NO: 197) TATCAGCCTTGTTTTCTCCCTCATTACTTGAAGGATATGAAGCTA AAACCCTTTTTTATAAAGCATTTGTCCGAATTCGGACATAATCA AAAAAGCTTAATTAAGATCAATTTGATCTACATCTCTTTAACCA ACAATATGTAAGATCTCAACTATCGCATCCGTGGATTAATTCAA TTATAACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACTGTA GAGGGGAGCACATTGATGCGAATTCATTAAAGAGGAGAAAGGT ACC dps TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTAT (SEQ ID CAATATATCTAACTCATTGAATCTTTATTAGTTTTGTTTTTCACG NO: 198) CTTGTTACCACTATTAGTGTGATAGGAACAGCCAGAATAGCGGA ACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGACA TAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGAGA AAGGTACC ahpC GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATC (SEQ ID CATGTCGTTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGG NO: 199) CAGGCACTGAAGATACCAAAGGGTAGTTCAGATTACACGGTCA CCTGGAAAGGGGGCCATTTTACTTTTTATCGCCGCTGGCGGTGC AAAGTTCACAAAGTTGTCTTACGAAGGTTGTAAGGTAAAACTTA TCGATTTGATAATGGAAACGCATTAGCCGAATCGGCAAAAATTG GTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATGCG AATTCATTAAAGAGGAGAAAGGTACC oxyS CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGCG (SEQ ID ATAGGTAGAATAGCAATGAACGATTATCCCTATCAAGCATTCTG NO: 200) ACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGTA CC

In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is deleted or mutated to reduce or eliminate wild-type activity.

In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a ROS-sensing transcription factor, e.g., the soxR gene, and a corresponding regulatory region, e.g., a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS-responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g., OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of ROS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the ROS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of ROS.

In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a payload(s). In some embodiments, the gene(s) capable of producing a payload(s) is present on a plasmid and operatively linked to a ROS-responsive regulatory region. In some embodiments, the gene(s) capable of producing a payload is present in a chromosome and operatively linked to a ROS-responsive regulatory region.

Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more payloads under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor.

In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a payload, such that the payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the payload. In some embodiments, the gene encoding the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the payload is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the payload. In some embodiments, the gene encoding the payload is expressed on a chromosome.

Propionate and Other Promoters

In some embodiments, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule expressed under the control of an inducible promoter that is responsive to specific molecules or metabolites in the environment, e.g., the mammalian gut. For example, the short-chain fatty acid propionate is a major microbial fermentation metabolite localized to the gut (Hosseini et al., 2011). In one embodiment, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is under the control of a propionate-inducible promoter. In a more specific embodiment, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is under the control of a propionate-inducible promoter that is activated by the presence of propionate in the mammalian gut. Any molecule or metabolite found in the mammalian gut, in a healthy and/or disease state, may be used to induce payload expression. Non-limiting examples of inducers include propionate, bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma glutamyl transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti-mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, and manganese. In alternate embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule and/or immune modulator is under the control of a pBAD promoter, which is activated in the presence of the sugar arabinose.

In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a plasmid and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a chromosome and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule, such that the gene or gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, a bacterium may comprise multiple copies of the gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule. In some embodiments, gene or gene cassette for producing the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing gene or gene cassette expression. In some embodiments, gene or gene cassette for producing the metabolic and/or satiety effector and/or immune modulator molecule is expressed on a chromosome.

Table 23 lists a propionate promoter sequence. In some embodiments, the propionate promoter is induced in the mammalian gut.

TABLE 23 Propionate promoter sequence Description Sequence Prp (Propionate) TTACCCGTCTGGATTTTCAGTACGCGCTTTTAAACGACGCCA promoter CAGCGTGGTACGGCTGATCCCCAAATAACGTGCGGCGGCGCG Bold: prpR CTTATCGCCATTAAAGCGTGCGAGCACCTCCTGCAATGGAAG Lower case: CGCTTCTGCTGACGAGGGCGTGATTTCTGCTGTGGTCCCCAC ribosome binding CAGTTCAGGTAATAATTGCCGCATAAATTGTCTGTCCAGTGT site TGGTGCGGGATCGACGCTTAAAAAAAGCGCCAGGCGTTCCAT ATG underlined: CATATTCCGCAGTTCGCGAATATTACCGGGCCAATGATAGTT start of gene of CAGTAGAAGCGGCTGACACTGCGTCAGCCCATGACGCACCGA interest TTCGGTAAAAGGGATCTCCATCGCGGCCAGCGATTGTTTTAA SEQ ID NO: 201 AAAGTTTTCCGCCAGAGGCAGAATATCAGGCTGTCGCTCGCG CAAGGGGGGAAGCGGCAGACGCAGAATGCTCAAACGGTAAAA CAGATCGGTACGAAAACGTCCTTGCGTTATCTCCCGATCCAG ATCGCAATGCGTGGCGCTGATCACCCGGACATCTACCGGGAT CGGCTGATGCCCGCCAACGCGGGTGACGGCTTTTTCCTCCAG TACGCGTAGAAGGCGGGTTTGTAACGGCAGCGGCATTTCGCC AATTTCGTCAAGAAACAGCGTGCCGCCGTGGGCGACCTCAAA CAGCCCCGCACGTCCACCTCGTCTTGAGCCGGTAAACGCTCC CTCCTCATAGCCAAACAGTTCAGCCTCCAGCAACGACTCGGT AATCGCGCCGCAATTAACGGCGACAAAGGGCGGAGAAGGCTT GTTCTGACGGTGGGGCTGACGGTTAAACAACGCCTGATGAAT CGCTTGCGCCGCCAGCTCTTTCCCGGTCCCTGTTTCCCCCTG AATCAGCACTGCCGCGCGGGAACGGGCATAGAGTGTAATCGT ATGGCGAACCTGCTCCATTTGTGGTGAATCGCCGAGGATATC GCTCAGCGCATAACGGGTCTGTAATCCCTTGCTGGAGGTATG CTGGCTATACTGACGCCGTGTCAGGCGGGTCATATCCAGCGC ATCATGGAAAGCCTGACGTACGGTGGCCGCTGAATAAATAAA GATGGCGGTCATTCCTGCCTCTTCCGCCAGGTCGGTAATTAG TCCTGCCCCAATTACAGCCTCAATGCCGTTAGCTTTGAGCTC GTTAATTTGCCCGCGAGCATCCTCTTCAGTGATATAGCTTCG CTGTTCAAGACGGAGGTGAAACGTTTTCTGAAAGGCGACCAG AGCCGGAATGGTCTCCTGATAGGTCACGATTCCCATTGAGGA AGTCAGCTTTCCCGCTTTTGCCAGAGCCTGTAATACATCGAA TCCGCTGGGTTTGATGAGGATGACAGGTACCGACAGTCGGCT TTTTAAATAAGCGCCGTTGGAACCTGCCGCGATAATCGCGTC GCAGCGTTCGGTTGCCAGTTTTTTGCGAATGTAGGCTACTGC CTTTTCAAAACCGAGCTGAATAGGCGTGATCGTCGCCAGATG ATCAAACTCCAGGCTGATATCCCGAAATAGTTCGAACAGGCG CGTTACCGAGACCGTCCAGATCACCGGTTTATCGCTATTATC GCGCGAAGCGCTATGCACAGTAACCATCGTCGTAGATTCATG TTTAAGGAACGAATTCTTGTTTTATAGATGTTTCGTTAATGT TGCAATGAAACACAGGCCTCCGTTTCATGAAACGTTAGCTGA CTCGTTTTTCTTGTGACTCGTCTGTCAGTATTAAAAAAGATT TTTCATTTAACTGATTGTTTTTAAATTGAATTTTATTTAATG GTTTCTCGGTTTTTGGGTCTGGCATATCCCTTGCTTTAATGA GTGCATCTTAATTAACAATTCAATAACAAGAGGGCTGAATag taatttcaacaaaataacgagcattcgaatg

Other Inducible Promoters

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an arabinose inducible system. The genes of arabinose metabolism are organized in one operon, AraBAD, which is controlled by the PAraBAD promoter. The PAraBAD (or Para) promoter suitably fulfills the criteria of inducible expression systems. PAraBAD displays tighter control of payload gene expression than many other systems, likely due to the dual regulatory role of AraC, which functions both as an inducer and as a repressor. Additionally, the level of ParaBAD-based expression can be modulated over a wide range of L-arabinose concentrations to fine-tune levels of expression of the payload. However, the cell population exposed to sub-saturating L-arabinose concentrations is divided into two subpopulations of induced and uninduced cells, which is determined by the differences between individual cells in the availability of L-arabinose transporter (Zhang et al., Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum; Appl. Environ. Microbiol. August 2012 vol. 78 no. 16 5831-5838). Alternatively, inducible expression from the ParaBad can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. An exemplary construct is depicted in FIG. 79.

In one embodiment, expression of one or more protein(s) of interest, e.g., one or more therapeutic polypeptide(s), is driven directly or indirectly by one or more arabinose inducible promoter(s).

In some embodiments, the arabinose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., arabinose.

In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more arabinose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the arabinose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., arabinose. In some embodiments, the cultures, which are induced by arabinose, are grown arerobically. In some embodiments, the cultures, which are induced by arabinose, are grown anaerobically.

In one embodiment, the arabinose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the arabinose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including arabinose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more arabinose promoters drive expression of one or more protein(s) of interest, in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, one or more protein(s) of interest are knocked into the arabinose operon and are driven by the native arabinose inducible promoter

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 202. In some embodiments, the arabinose inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 203. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 204.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a rhamnose inducible system. The genes rhaBAD are organized in one operon which is controlled by the rhaP BAD promoter. The rhaP BAD promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which divergently transcribed in the opposite direction of rhaBAD. In the presence of L-rhamnose, RhaR binds to the rhaP RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose then bind to the rhaP BAD and the rhaP T promoter and activate the transcription of the structural genes. In contrast to the arabinose system, in which AraC is provided and divergently transcribed in the gene sequence(s), it is not necessary to express the regulatory proteins in larger quantities in the rhamnose expression system because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression. Alternatively, inducible expression from the rhaBAD can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. An exemplary construct is depicted in FIG. 82B (construct for PAL expression under the control of a rhamnose inducible promoter).

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s). In one embodiment, expression of the payload is driven directly or indirectly by a rhamnose inducible promoter.

In some embodiments, the rhamnose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., rhamnose

In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more rhamnose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the rhamnose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., rhamnose. In some embodiments, the cultures, which are induced by rhamnose, are grown arerobically. In some embodiments, the cultures, which are induced by rhamnose, are grown anaerobically.

In one embodiment, the rhamnose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the rhamnose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., rhamnose and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including rhamnose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more rhamnose promoters drive expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 205.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible system or other compound which induced transcription from the Lac Promoter. IPTG is a molecular mimic of allolactose, a lactose metabolite that activates transcription of the lac operon. In contrast to allolactose, the sulfur atom in IPTG creates a non-hydrolyzable chemical blond, which prevents the degradation of IPTG, allowing the concentration to remain constant. IPTG binds to the lac repressor and releases the tetrameric repressor (lacI) from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. Since IPTG is not metabolized by E. coli , its concentration stays constant and the rate of expression of Lac promoter-controlled is tightly controlled, both in vivo and in vitro. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved. Inducible expression from the PLac can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. Other compounds which inactivate LacI, can be used instead of IPTG in a similar manner.

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s).

In some embodiments, the IPTG inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., IPTG.

In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the IPTG inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., IPTG. In some embodiments, the cultures, which are induced by IPTG, are grown arerobically. In some embodiments, the cultures, which are induced by IPTG, are grown anaerobically.

In one embodiment, the IPTG inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the IPTG inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including IPTG presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more IPTG inducible promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 206. In some embodiments, the IPTG inducible construct further comprises a gene encoding lacI, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 207. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 208.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a tetracycline inducible system. The initial system Gossen and Bujard (Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen M & Bujard H. PNAS. 1992 Jun. 15; 89(12):5547-51) developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. Tetracycline-controlled transactivator (tTA) was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16) from herpes simplex virus.In the absence of tetracycline, the tetR portion of tTA will bind tetO sequences in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, precluding tTA from binding to the tetO sequences. Next, a reverse Tet repressor (rTetR), was developed which created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA (reverse tetracycline-controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system.

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a tetracycline inducible promoter.

In some embodiments, the tetracycline inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, is driven directly or indirectly by one or more tetracycline inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., tetracycline

In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the tetracycline inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., tetracycline. In some embodiments, the cultures, which are induced by tetracycline, are grown arerobically. In some embodiments, the cultures, which are induced by tetracycline, are grown anaerobically.

In one embodiment, the tetracycline inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the tetracycline inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., tetracycline and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including tetracycline presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more tetracycline promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the bolded sequences of SEQ ID NO: 213 (tet promoter is in bold). In some embodiments, the tetracycline inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 213 in italics (Tet repressor is in italics). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 213 in italics (Tet repressor is in italics).

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) whose expression is controlled by a temperature sensitive mechanism. Thermoregulators are advantageous because of strong transcriptional control without the use of external chemicals or specialized media (see, e.g., Nemani et al., Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy; J Biotechnol. 2015 Jun. 10; 203: 32-40, and references therein). Thermoregulated protein expression using the mutant c1857 repressor and the pL and/or pR phage promoters have been used to engineer recombinant bacterial strains. The gene of interest cloned downstream of the 2\, promoters can then be efficiently regulated by the mutant thermolabile c1857 repressor of bacteriophage λ. At temperatures below 37° C., cI857 binds to the oL or oR regions of the pR promoter and blocks transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. An exemplary construct is depicted in FIG. 82A. Inducible expression from the ParaBad can be controlled or further fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.

In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a thermoregulated promoter.

In some embodiments, the thermoregulated promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., temperature.

In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, it may be advantageous to shup off production of the one or more protein(s) of interest. This can be done in a thermoregulated system by growing the strain at lower temperatures, e.g., 30 C. Expression can then be induced by elevating the temperature to 37 C and/or 42 C. In some embodiments, the thermoregulated promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the cultures, which are induced by temperatures between 37 C and 42 C, are grown arerobically. In some embodiments, the cultures, which are induced by induced by temperatures between 37 C and 42 C, are grown anaerobically.

In one embodiment, the thermoregulated promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabino se). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., permissive temperature, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 209. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest . In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 210. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 212.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are indirectly inducible through a system driven by the PssB promoter. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions.

This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. As a result, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. FIG. 83A depicts a schematic of the gene organization of a PssB promoter.

In one embodiment, expression of one or more protein(s) of interest is indirectly regulated by a repressor expressed under the control of one or more PssB promoter(s).

In some embodiments, induction of the RssB promoter(s) indirectly drives the in vivo expression of one or more protein(s) of interest. In some embodiments, induction of the RssB promoter(s) indirectly drives the expression of one or more protein(s) of interest during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, conditions for induction of the RssB promoter(s) are provided in culture, e.g., in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.

In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

In another non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 214.

Sequences useful for expression from inducible promoters are listed in Table 24.

TABLE 24 Inducible promoter construct sequences Description Sequence Arabinose CAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGC Promoter region TAACCCAACCGGTAACCCCGCTTATTAAAAGCATTCTGTA SEQ ID NO: ACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAA 202 AAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTAT TTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATC CATAAGATTAGCGGATCCAGCCTGACGCTTTTTTTCGCAA CTCTCTACTGTTTCTCCATACCTCTAGAAATAATTTTGTTT AACTTTAAGAAGGAGATATACAT AraC (reverse TTATTCACAACCTGCCCTAAACTCGCTCGGACTCGCCCCG orientation) GTGCATTTTTTAAATACTCGCGAGAAATAGAGTTGATCGT SEQ ID NO: CAAAACCGACATTGCGACCGACGGTGGCGATAGGCATCC 203 GGGTGGTGCTCAAAAGCAGCTTCGCCTGACTGATGCGCTG GTCCTCGCGCCAGCTTAATACGCTAATCCCTAACTGCTGG CGGAACAAATGCGACAGACGCGACGGCGACAGGCAGACA TGCTGTGCGACGCTGGCGATATCAAAATTACTGTCTGCCA GGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATT ATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATG CGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCA ATTCCGAATAGCGCCCTTCCCCTTGTCCGGCATTAATGATT TGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCAT CCGGGCGAAAGAAACCGGTATTGGCAAATATCGACGGCC AGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGT AAACCCACTGGTGATACCATTCGTGAGCCTCCGGATGACG ACCGTAGTGATGAATCTCTCCAGGCGGGAACAGCAAAAT ATCACCCGGTCGGCAGACAAATTCTCGTCCCTGATTTTTCA CCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACC TTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAA CCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGG CGTTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGC TTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAA ACCAATTGTCCATATTGCAT AraC MQYGQLVSSLNGGSMKSMAEAQNDPLLPGYSFNAHLVAGL polypeptide TPIEANGYLDFFIDRPLGMKGYILNLTIRGQGVVKNQGREFV SEQ ID NO: CRPGDILLFPPGEIHHYGRHPEAHEWYHQWVYFRPRAYWHE 204 WLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYS ELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHL ADSNFDIASVAQHVCLSPSRLSHLFRQQLGISVLSWREDQRIS QAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSE FRAGCE* Region CGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAAC comprising ATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTT rhamnose TCCTGTCAGTAACGAGAAGGTCGCGAATCAGGCGCTTTTT inducible AGACTGGTCGTAATGAAATTCAGCTGTCACCGGATGTGCT promoter TTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTAC SEQ ID NO: AAATAATTTTGTTTAAAACAACACCCACTAAGATAACTCT 205 AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT Lac Promoter ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATG region CCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCG SEQ ID NO: CTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGA 206 TCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATG TGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATG TGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCT CTACAAATAATTTTGTTTAAAACAACACCCACTAAGATAA CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA CAT LacO GGAATTGTGAGCGCTCACAATT LacI (in reverse TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC orientation) TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT SEQ ID NO: GCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA 207 GACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGA GAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCA GGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATA ACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAG ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCAT CGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTT TGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTT CCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATG CCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAA TGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG ACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGG AGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATC AAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCAC AGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATC AGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCG CTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCG CCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGG AGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAG TTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCC ATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTG GCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAG ACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTT TCAT LacI MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEA polypeptide AMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAA sequence IKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLI SEQ ID NO: INYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT 208 RLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRN QIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQ MALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIK QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLA PNTQTASPRALADSLMQLARQVSRLESGQ Region ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACC comprising GTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTG Temperature CATAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCC sensitive GTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAA promoter CAACACCCACTAAGATAACTCTAGAAATAATTTTGTTTAA SEQ ID NO: CTTTAAGAAGGAGATATACAT 209 mutant cI857 TCAGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACT repressor TTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTG SEQ ID NO: GGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCT 210 ATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCA AGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCT CAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGG CTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACC TCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGC TTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGC TTAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACA GGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAA CCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGG GCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGTAAGCA ATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAA TAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCT GCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTT TTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGC TCTTGTGTTAATGGTTTCTTTTTTGTGCTCAT RBS and leader CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA region CAT SEQ ID NO: 211 mutant cI857 MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKM repressor GMGQSGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAR polypeptide EIYEMYEAVSMQPSLRSEYEYPVFSHVQAGMFSPKLRTFTKG sequence DAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGML SEQ ID NO: ILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNP 212 QYPMIPCNESCSVVGKVIASQWPEETFG TetR-Tet Ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataa promoter gaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcata construct ctatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacct SEQ ID NO: aaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttgg 213 cataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgta cttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaat cttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggct aaggcgtcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacaccta gcttctgggcgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcg ctgttaatcactttacttttatctaatctagacatcattaattcctaatttttgttgacactctatcattg atagagttattttaccactccctatcagtgatagagaaaagtgaa ctctagaaataattttgttt aactttaagaaggagatatacat PssB promoter tcacctttcccggattaaacgcttttttgcccggtggcatggtgctaccggcgatcacaaacggtta SEQ ID NO: attatgacacaaattgacctgaatgaatatacagtattggaatgcattacccggagtgttgtgtaac 214 aatgtctggccaggtttgtttcccggaaccgaggtcacaacatagtaaaagcgctattggtaatgg tacaatcgcgcgtttacacttattc

Constitutive Promoters

In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter.

In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is active under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the constitutive promoteris active under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.

In some embodiments, the constitutive promoterthat is operably linked to the gene encoding the payload is active in various exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions).

In some embodiments, the constitutive promoteris active in exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the constitutive promoteris active in exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the constitutive promoteris active in low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the constitutive promoteris active in the presence of molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the constitutive promoteris directly or indirectly induced by a molecule that is co-administered with the bacterial cell. In some embodiments, the constitutive promoteris active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions.

Bacterial constitutive promoters are known in the art. For example, a lisitng of suitable promoters from a number of bacterial species and bacteriophages can be found at: http://parts.igem.org/Promoters/Catalog/Constitutive.

Induction of Payloads During Strain Culture

In some embodiments, it is desirable to pre-induce payload or protein of interest expression and/or payload activity prior to administration. Such payload or protein of interest may be an effector intended for secretion or may be an enzyme which catalyzes a metabolic reaction to produce an effector. In other embodiments, the protein of interest is an enzyme which catabolizes a harmful metabolite. In such situations, the strains are pre-loaded with active payload or protein of interest. In such instances, the genetically engineered bacteria of the invention express one or more protein(s) of interest, under conditions provided in bacterial culture during cell growth, expansion, purification, fermentation, and/or manufacture prior to administration in vivo. Such culture conditions can be provided in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. As used herein, the term “bacterial culture” or bacterial cell culture” or “culture” refers to bacterial cells or microorganisms, which are maintained or grown in vitro during several production processes, including cell growth, cell expansion, recovery, purification, fermentation, and/or manufacture. As used herein, the term “fermentation” refers to the growth, expansion, and maintenance of bacteria under defined conditions. Fermentation may occur under a number of cell culture conditions, including anaerobic or low oxygen or oxygenated conditions, in the presence of inducers, nutrients, at defined temperatures, and the like.

Culture conditions are selected to achieve optimal activity and viability of the cells, while maintaining a high cell density (high biomass) yield. A number of cell culture conditions and operating parameters are monitored and adjusted to achieve optimal activity, high yield and high viability, including oxygen levels (e.g., low oxygen, microaerobic, aerobic), temperature of the medium, and nutrients and/or different growth media, chemical and/or nutritional inducers and other components provided in the medium. In some embodiments, phenylalanine is added to the media, e.g., to boost cell health. Without wishing to be bound by theory, addition of phenylalanine to the medium may prevent bacteria from catabolizing endogenously produced phenylalanine required for cell growth.

In some embodiments, the one or more protein(s) of interest and are directly or indirectly induced, while the strains is grown up for in vivo administration. Without wishing to be bound by theory, pre-induction may boost in vivo activity. This is particularly important in proximal regions of the gut which are reached first by the bacteria, e.g., the small intestine. If the bacterial residence time in this compartment is relatively short, the bacteria may pass through the small intestine without reaching full in vivo induction capacity. In contrast, if a strain is pre-induced and preloaded, the strains are already fully active, allowing for greater activity more quickly as the bacteria reach the intestine. Ergo, no transit time is “wasted”, in which the strain is not optimally active. As the bacteria continue to move through the intestine, in vivo induction occurs under environmental conditions of the gut (e.g., low oxygen, or in the presence of gut metabolites).

In one embodiment, expression of one or more payload(s), is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of several different proteins of interest is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), is driven from the same promoter as a multicistronic message. In one embodiment, expression of one or more payload(s) is driven from the same promoter as two or more separate messages. In one embodiment, expression of one or more payload(s) is driven from the one or more different promoters.

In some embodiments, the strains are administered without any pre-induction protocols during strain growth prior to in vivo administration.

Anaerobic Induction

In some embodiments, cells are induced under anaerobic or low oxygen conditions in culture. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and exponential growth and are then switched to anaerobic or low oxygen conditions for approximately 3 to 5 hours. In some embodiments, strains are induced under anaerobic or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more payload(s) and /or Phe transporters under the control of one or more FNR promoters.

In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. In one embodiment, expression of several different proteins of interest is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions.

In one embodiment, expression of two or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under anaerobic or low oxygen conditions.

Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under anaerobic or low oxygen culture conditions, and in vivo, under the low oxygen conditions found in the gut.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under anaerobic or low oxygen conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

In one embodiment, expression of one or more Payload is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. In one embodiment, the chemical and/or nutritional inducer is arabino se and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under anaerobic and/or low oxygen conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under anaerobic or low oxygen conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload and or Phe transporter sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Aerobic Induction

In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic conditions. This allows more efficient growth and viability, and, in some cases, reduces the build-up of toxic metabolites. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature, for approximately 3 to 5 hours.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art can be induced under aerobic conditions in the presence of the chemical and/or nutritional inducer during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under aerobic conditions.

In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

In some embodiments, promoters regulated by temperature are induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s)and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the one or more different promoters under aerobic conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced under aerobic conditions. In some embodiments, a strain comprises three or more different promoters which are induced under aerobic culture conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. a chemically inducible promoter, and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter under aerobic culture conditions. In some embodiments two or more chemically induced promoter gene sequence(s) are combined with a thermoregulated construct described herein. In one embodiment, the chemical and/or nutritional inducer is arabino se and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload and or Phe transporter sequence(s) under the control of one or more constitutive promoter(s) active under aerobic conditions.

In some embodiments, genetically engineered strains comprise gene sequence(s) which are induced under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

In some embodiments, genetically engineered strains comprise gene sequence(s), which are arabinose inducible under aerobic culture conditions. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

In some embodiments, genetically engineered strains comprise gene sequence(s), which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

In some embodiments, genetically engineered strains comprise gene sequence(s) which are arabinose inducible under aerobic culture conditions. In some embodiments, such a strain further comprises sequence(s) which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene payload and/or Phe transporter sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

As evident from the above non-limiting examples, genetically engineered strains comprise inducible gene sequence(s) which can be induced numerous combinations. For example, rhamnose or tetracycline can be used as an inducer with the appropriate promoters in addition or in lieu of arabinose and/or IPTG or with thermoregulation. Additionally, such bacterial strains can also be induced with the chemical and/or nutritional inducers under anaerobic conditions.

Microaerobic Induction

In some embodiments, viability, growth, and activity are optimized by pre-inducing the bacterial strain under microaerobic conditions. In some embodiments, microaerobic conditions are best suited to “strike a balance” between optimal growth, activity and viability conditions and optimal conditions for induction; in particular, if the expression of the one or more payload(s) and/or Phe transporter(s) are driven by a anaerobic and/or low oxygen promoter, e.g., a FNR promoter. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to at a permissive temperature, for approximately 3 to 5 hours.

In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under microaerobic conditions.

Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under microaerobic culture conditions, and in vivo, under the low oxygen conditions found in the gut.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under microaerobic conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under microaerobic conditions.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under microaerobic conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g., an anaerobic/low oxygen promoter or microaerobic promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen or microaerobic promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Induction of Strains Using Phasing, Pulsing and/or Cycling

In some embodiments, cycling, phasing, or pulsing techniques are emplyed during cell growth, expansion, recovery, purification, fermentation, and/or manufacture to efficienty induce and grow the strains prior to in vivo administration. This method is used to “strike a balance” between optimal growth, activity, cell health, and viability conditions and optimal conditions for induction; in particular, if growth, cell health or viability are negatively affected under inducing conditions. In such instances, cells are grown (e.g., for 1.5 to 3 hours) in a first phase or cycle until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10̂8 to 1×10̂11, and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature (if a promoter is thermoregulated), or change in oxygen levels (e.g., reduction of oxygen level in the case of induction of an FNR promoter driven construct) for approximately 3 to 5 hours. In a second phase or cycle, conditions are brought back to the original conditions which support optimal growth, cell health and viability. Alternatively, if a chemical and/or nutritional inducer is used, then the culture can be spiked with a second dose of the inducer in the second phase or cycle.

In some embodiments, two cycles of optimal conditions and inducing conditions are employed (i.e, growth, induction, recovery and growth, induction). In some embodiments, three cycles of optimal conditions and inducing conditions are employed. In some embodiments, four or more cycles of optimal conditions and inducing conditions are employed. In a non-liming example, such cycling and/or phasing is used for induction under anaerobic and/or low oxygen conditions (e.g., induction of FNR promoters). In one embodiment, cells are grown to the optimal density and then induced under anaerobic and/or low oxygen conditions. Before growth and/or viability are negatively impacted due to stressful induction conditions, cells are returned to oxygenated conditions to recover, after which they are then returned to inducing anaerobic and/or low oxygen conditions for a second time. In some embodiments, these cycles are repeated as needed.

In some embodiments, growing cultures are spiked once with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked twice with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked three or more times with the chemical and/or nutritional inducer. In a non-limiting example, cells are first grown under optimal growth conditions up to a certain density, e.g., for 1.5 to 3 hour) to reached an of 0.1 to 10, until the cells are at a density ranging from 1×10̂8 to 1×10̂11. Then the chemical inducer, e.g., arabinose or IPTG, is added to the culture. After 3 to 5 hours, an additional dose of the inducer is added to re-initiate the induction. Spiking can be repeated as needed.

In some embodiments, phasing or cycling changes in temperature in the culture. In another embodiment, adjustment of temperature may be used to improve the activity of a payload. For example, lowering the temperature during culture may improve the proper folding of the payload. In such instances, cells are first grown at a temperature optimal for growth (e.g., 37 C). In some embodiments, the cells are then induced, e.g., by a chemical inducer, to express the payload. Concurrently or after a set amount of induction time, the temperature in the media is lowered, e.g., between 25 and 35 C, to allow improved folding of the expressed payload, e.g., PAL.

In some embodiments, payload(s) are under the control of different inducible promoters, for example two different chemical inducers. In other embodiments, the payload is induced under low oxygen conditions or microaerobic conditions and a second payload is induced by a chemical inducer.

In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture by using phasing or cycling or pulsing or spiking techniques.

In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters through the employment of phasing or cycling or pulsing or spiking techniques.

In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced through the employment of phasing or cycling or pulsing or spiking techniques in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) and/or Phe transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more constitutive promoter(s) described herein and are induced through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein, and are induced through the employment of phasing or cycling or pulsing or spiking techniques.

Any of the strains described herein can be grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions.

In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message and which are induced through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages and is grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters, all of which are induced through the employment of phasing or cycling or pulsing or spiking techniques.

In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions. Any of the strains described in these embodiments may be induced through the employment of phasing or cycling or pulsing or spiking techniques.

Aerobic Induction of the FNR Promoter

FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis A J The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In some embodiments, oxygen bypass system shown and described in FIG. 78 is used. In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of the protein of interest (e.g., one or more metabolic effector(s) described herein) by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of the protein of interest. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the protein of interest, e.g., one or more metabolic effector(s) described herein.

In some embodiments, FNRS24Y is expressed during aerobic culture growth and induces a gene of interest. In other embodiments described herein, a second payload expression can also be induced aerobically, e.g., by arabinose. In a non-limiting example, a protein of interest and FNRS24Y can in some embodiments be induced simultaneously, e.g., from an arabinose inducible promoter. In some embodiments, FNRS24Y and the protein of interest (e.g., one or more metabolic effector(s) described herein) are transcribed as a bicistronic message whose expression is driven by an arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression to be driven from the endogenous Para promoter.

In some embodiments, a LacI promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

Mutagenesis

In some embodiments, an inducible promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, an oxygen level-dependent promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, the oxygen level-dependent promoter is mutagenized, and mutants are selected based upon the level of detectable product, e.g., by flow cytometry, fluorescence-activated cell sorting (FACS) when the detectable product fluoresces. In some embodiments, one or more transcription factor binding sites is mutagenized to increase or decrease binding. In alternate embodiments, the wild-type binding sites are left intact and the remainder of the regulatory region is subjected to mutagenesis. In some embodiments, the mutant promoter is inserted into the genetically engineered bacteria of the invention to increase expression of the metabolic and/or satiety effector and/or immune modulator molecule in low-oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the oxygen level-sensing transcription factor and/or the oxygen level-dependent promoter is a synthetic, non-naturally occurring sequence. In some embodiments, the transcription factor regulating the mutated promoter senses the presence of certain molecules or metabolites, the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, the gene encoding a metabolic and/or satiety effector and/or immune modulator molecule is mutated to increase expression and/or stability of said molecule in low oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, one or more of the genes in a gene cassette for producing a metabolic and/or satiety effector and/or immune modulator molecule is mutated to increase expression of said molecule in low oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions.

Multiple Mechanisms of Action

In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/1, araC/BAD, lacZ, dapA, cea, and other shown in FIG. 57. For example, the genetically engineered bacteria may include four copies of GLP-1 inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ. Alternatively, the genetically engineered bacteria may include three copies of GLP-1 inserted at three different insertion sites, e.g., malE/K, insB/I, and lacZ, and three copies of a butyrogenic gene cassette inserted at three different insertion sites, e.g., dapA, cea, and araC/BAD.

In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at three different insertion sites and three copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at three different insertion sites.

In some embodiments, the genetically engineered bacteria comprise one or more of (1) one or more gene(s) or gene cassette(s) for the production of propionate, as described herein (2) one or more gene(s) or gene cassette(s) for the production of butyrate, as described herein (3) one or more gene(s) or gene cassette(s) for the production of acetate, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more of GLP-1 and GLP-1 analogs, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more bile salt hydrolases, as described herein (5) one or more gene(s) or gene cassette(s) for the production of one or more transporters, e.g. for the import of bile salts and/or metabolites, e.g. tryptophan and/or tryptophan metabolites, as described herein (6) one or more polypetides for secretion, including but not limited to.GLP-1 and its analogs, bile salt hydrolases, and tryptophan synthesis and/or catabolic enzymes of the tryptophan degradation pathways, in wild type or in mutated form (for increased stability or metabolic activity) (3) one or more components of secretion machinery, as described herein (4) one or more auxotrophies, e.g., deltaThyA (5) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance (6) one or more mutations/deletions to increase the flux through a metabolic pathway encoded by one or more genes or gene cassette(s), e.g mutations/deletions in genes in NADH consuming pathways, genes involved in feedback inhibition of a metabolic pathway encoded by the gene(s) or gene cassette(s) genes, as described herein (7) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway.

In some embodiments, under conditions where the gene, gene(s), or gene cassettes for producing the payload(s) is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the payload(s) as compared to unmodified bacteria of the same subtype under the same conditions.

In some embodiments, the genetically engineered bacteria of the invention produce at least one metabolic and/or satiety effector and/or immune modulator molecule under inducing conditions and are capable of reducing one or more symptoms of metabolic disease in a subject by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to unmodified bacteria of the same subtype under the same conditions. In some embodiments, under conditions where the payload is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the payload, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.

Symptoms and manifestations of metabolic diseases may be measured by methods known in the art, e.g., glucose tolerance testing, insulin tolerance testing.

In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of a metabolic and/or satiety effector and/or immune modulator molecule under inducing conditions than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the metabolic and/or satiety effector and/or immune modulator molecule. In embodiments using genetically modified forms of these bacteria, the metabolic and/or satiety effector and/or immune modulator molecule will be detectable under inducing conditions.

In certain embodiments, the metabolic and/or satiety effector and/or immune modulator molecule is butyrate. Methods of measuring butyrate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 μM/OD, at least about 10 μM/OD, at least about 100 μM/OD, at least about 500 μM/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In certain embodiments, the metabolic and/or satiety effector and/or immune modulator molecule is propionate. Methods of measuring propionate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Hillman 1978; Lukovac et al., 2014). In some embodiments, measuring the activity and/or expression of one or more gene products in the propionate gene cassette serves as a proxy measurement for propionate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure propionate production. In alternate embodiments, propionate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 μM, at least about 10 μM, at least about 100 μM, at least about 500 μM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, or at least about 50 mM of propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the gene, gene(s), or gene cassettes for producing the payload(s). Primers may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload RNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C., 60-70° C., and 30-50° C. for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C., 55-65° C., and 35-45° C. for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload(s).

In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the payload(s). Primers may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C., 60-70° C., and 30-50° C. for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C., 55-65° C., and 35-45° C. for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload(s).

Secretion

In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism or non-native secretion mechanism that is capable of secreting a molecule from the bacterial cytoplasm in the extracellular environment. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane.

In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane-spanning secretion system. Membrane-spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in FIG. 61, FIG. 62, FIG. 63, FIG. 64, FIG. 65, FIG. 66, and FIG. 67. Mycobacteria, which have a Gram-negative-like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al., 2003). With the exception of the T2SS, double membrane-spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane-spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane-spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system or autosecreter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015).

In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type III-like secretion system (T3SS) from Shigella, Salmonella, E. coli , Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. Thus, the molecule is secreted into the gut lumen or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the molecule of interest from the bacterial cytoplasm. In some embodiments, the secreted molecule, such as a heterologouse protein or peptide comprises a type III secretion sequence that allows the molecule of interest o be secreted from the bacteria.

In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment. For example, a modified flagellar type III secretion apparatus in which untranslated DNA fragment upstream of the gene fliC (encoding flagellin), e.g., a 173-bp region, is fused to the gene encoding the polypeptide of interest can be used to secrete heterologous polypeptides (See, e.g., Majander et al., Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol. 2005 Apr.; 23(4):475-81). In some cases, the untranslated region from the fliC loci, may not be sufficient to mediate translocation of the passenger peptide through the flagella. Here it may be necessary to extend the N-terminal signal into the amino acid coding sequence of FliC, for example using the 173 bp of untranslated region along with the first 20 amino acids of FliC (see, e.g., Duan et al., Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes, Appl. Environ. Microbiol. December 2008 vol. 74 no. 23 7437-7438).

In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in FIG. 62, a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. The N-terminal, Sec-dependent signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex (Teta-barrel assembly machinery') where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is threaded through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once exposed to the extracellular environment, the therapeutic peptide can be freed from the linker system by an autocatalytic cleavage (left side of Bam complex) or by targeting of a membrane-associated peptidase (black scissors; right side of Bam complex) to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises an N-terminal secretion signal, a linker, and beta-domain of an autotransporter so as to allow the molecule to be secreted from the bacteria.

In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. FIG. 63 shows the alpha-hemolysin (HlyA) of uropathogenic Escherichia coli. This pathway uses HlyB, an ATP-binding cassette transporter; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane-spanning secretion system. Single membrane-spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g., the SecYEG complex in E. coli ), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g., Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii, Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the molecule of interest from the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads.

In order to translocate a protein, e.g., therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g., therapeutic polypeptides)—particularly those of eukaryotic origin—contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.

One way to secrete properly folded proteins in gram-negative bacteria—particularly those requiring disulphide bonds—is to target the reducing-environment periplasm in conjunction with a destabilizing outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These “leaky” gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a “leaky” or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, 1 pp, ompC, ompA, ompF, tolA, to1B, pal, degS, degP, and nlpI. Lpp is the most abundant polypeptide in the bacterial cell existing at ˜500,000 copies per cell and functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. 1.

Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). To1A-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are inactivated. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpI can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated 1 pp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, to1B, and pal genes. in some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlp1. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from 1pp, ompA, ompF, to1 A, to1B, pal, degS, degP, and nlpl genes.

To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected from 1 pp, ompA, ompF, to1A, to1B, pa1, degS, degP, and nlp1, under the control of an inducible promoter. For example, expression of 1 pp or other cell wall stability protein or periplasmic protease can be repressed in conditions where the therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., over expression of colicins or the third topological domain of To1A, which peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.

The Table 25 and Table 26 below lists secretion systems for Gram positive bacteria and Gram negative bacteria.

TABLE 25 Secretion systems for gram positive bacteria Bacterial Strain Relevant Secretion System C. novyi-NT (Gram+) Sec pathway Twin-arginine (TAT) pathway C. butryicum (Gram+) Sec pathway Twin-arginine (TAT) pathway Listeria monocytogenes (Gram+) Sec pathway Twin-arginine (TAT) pathway

TABLE 26 Secretion Systems for Gram negative bacteria Protein secretary pathways (SP) in gram-negative bacteria and their descendants # Type Proteins/ Energy (Abbreviation) Name TC#² Bacteria Archaea Eukarya System Source IMPS - Gram-negative bacterial inner membrane channel-forming translocases ABC (SIP) ATP binding 3.A.1 + + + 3-4 ATP cassette translocase SEC (IISP) General secretory 3.A.5 + + + ~12 GTP OR translocase ATP + PMF Fla/Path (IIISP) Flagellum/ 3.A.6 + − − >10 ATP virulence- related translocase Conj (IVSP) Conjugation-related 3.A.7 + − − >10 ATP translocase Tat (IISP) Twin-arginine 2.A.64 + + + 2-4 PMF targeting (chloroplasts) translocase Oxa1 (YidC) Cytochrome oxidase 2.A.9 + + + 1 None biogenesis family (mitochondria or chloroplasts) PMF MscL Large conductance 1.A.22 + + + 1 None mechanosensitive channel family Holins Holin functional 1.E.1•21 + − − 1 None superfamily Eukaryotic Organelles MPT Mitochondria 1 3.A.B − − + >20 ATP protein translocase (mitochondrial) CEPT Chloroplast envelope 3.A.9 (+) − + ≥3 GTP protein translocase (chloroplasts) Bcl-2 Eukaryotic Bcl-2 1.A.21 − − + 1? None family (programmed cell death) Gram-negative bacterial outer membrane channel-forming translocases MTB (IISP) Main terminal 3.A.15 +^(b) − − ~14 ATP; branch of the PMF general secretory translocase FUP AT-1 Fimbrial 1.B.11 +^(b) − − 1 None usher protein 1.B.12 +^(b) − 1 None Autotransporter-1 AT-2 OMF (ISP) Autotransporter-2 1.B.40 +^(b) − − 1 None 1.B.17 +^(b) +(?) 1 None TPS Secretin 1.B.20 + − + 1 None (IISP and IISP) 1.B.22 +^(b) − 1 None OmpIP Outer membrane 1.B.33 + − + ≥4 None? insertion porin (mitochondria; chloroplasts)

The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other molecules from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe/Volume 1, Number 9, 2006 “Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently”, the contents of which is herein incorporated by reference in its entirety.

Any of the secretion systems described herein may according to the disclosure be employed to secrete the proteins of interest. Non-limiting examples of proteins of interest include GLP-1 peptides, GLP-1 analogs, GLP-2 peptides, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, tryptophan synthesis enzymes, SCFA biosynthesis enzymes, tryptophan catabolic enzymes, e.g. in the indole pathway and/or the kynurenine pathway, and bile salt hydrolases, as described herein. These polypeptides may be mutated to increase stability, resistance to protease digestion, and/or activity.

TABLE 27 Comparison of Secretion systems for secretion of polypeptide from engineered bacteria Secretion System Tag Cleavage Advantages Other features Modified mRNA No No peptide tag May not be as Type III (or N- cleavage Endogenous suited for larger (flagellar) terminal) necessary proteins Deletion of flagellar genes Type V N- and Yes Large proteins 2-step secretion autotransport C- Endogenous terminal Cleavable Type I C- No Tag; Exogenous terminal Machinery Diffusible N- Yes Disulfide bond May affect cell Outer terminal formation fragility/ Membrane survivability/ (DOM) growth/yield

In some embodiments, the therapeutic polypeptides of interest are secreted using components of the flagellar type III secretion system. In a non-limiting example, such a therapeutic polypeptide of interest, such as, one or more GLP-1 peptides, GLP-1 analogs, GLP-2 peptides, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, tryptophan synthesis enzymes, SCFA biosynthesis enzymes, tryptophan catabolic enzymes, and/or bile salt hydrolases, is assembled behind a fliC-5′UTR (e.g., 173-bp untranslated region from the fliC loci), and is driven by the native promoter. In other embodiments, the expression of the therapeutic peptide of interested secreted using components of the flagellar type III secretion system is driven by a tet-inducible promoter. In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose is used. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing conditions, as described herein and known in the art, can be used. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing and in vivo conditions, described herein and known in the art, can be used. In other embodiments, a constitutive promoter can be used. In some embodiments, the therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium copy plasmid). In some embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into fliC locus (thereby deleting fliC), where it is driven by the native FliC promoter. In some embodiments, an N terminal part of FliC (e.g., the first 20 amino acids of FliC) is included in the construct, to further increase secretion efficiency.

In some embodiments, the therapeutic polypeptides of interest, e.g., such as, one or more GLP-1 peptides, GLP-1 analogs, GLP-2 peptides, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, tryptophan synthesis enzymes, SCFA biosynthesis enzymes, and/or tryptophan catabolic enzymes, and/or bile salt hydrolases, are secreted using via a diffusible outer membrane (DOM) system. In some embodiments, the therapeutic polypeptide of interest is fused to a N-terminal Sec-dependent secretion signal. Non-limiting examples of such N-terminal Sec-dependent secretion signals include PhoA, OmpF, OmpA, and cvaC. In alternate embodiments, the therapeutic polypeptide of interest is fused to a Tat-dependent secretion signal. Exemplary Tat-dependent tags include TorA, FdnG, and DmsA. In some embodiments, expression of the secretion-tagged therapeutic protein is driven by a tet promoter or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), or by promoters induced by molecules specific to certain metabolic conditions, or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing conditions, as described herein and known in the art, can be used. Alternatively, a promoter that is inducible in vitro, e.g., under cell culture, cell production/maufacturing and in vivo conditions, described herein and known in the art, can be used. In other embodiments, a constitutive promoter can be used. In some embodiments, the secretion-tagged therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium copy plasmid). In other embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into the bacterial chromosome, e.g., at one or more of the integration sites shown in FIG. 57. In certain embodiments, the genetically engineered bacteria comprise deletions or mutations in one or more of the outer membrane and/or periplasmic proteins. Non-limiting examples of such proteins, one or more of which may be deleted or mutated, include 1pp, pal, to1A, and/or nlpI. In some embodiments, 1 pp is deleted or mutated. In some embodiments, pal is deleted or mutated. In some embodiments, to1 A is deleted or mutated. In other embodiments, nlpl is deleted or mutated. In yet other embodiments, certain periplasmic proteases are deleted or mutated, e.g., to increase stability of the polypeptide in the periplasm. Non-limiting examples of such proteases include degP and ompT. In some embodiments, degP is deleted or mutated. In some embodiments, ompT is deleted or mutated. In some embodiments, degP and ompT are deleted or mutated.

In some embodiments, the therapeutic polypeptides of interest, e.g., GLP-1, GLP-1 peptides, GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, SCFA producing enzymes, Tryptophan catabolism enzymes, and/or bile salt hydrolases are secreted via a Type V Auto-secreter (pic Protein) Secretion. In some embodimetns, the therapeutic protein of interest is expressed as a fusion protein with the native Nissle auto-secreter E. coli_01635 (where the original passenger protein is replaced with the therapeutic polypeptides of interest.

In some embodiments, the therapeutic polypeptides of interest, e.g., GLP-1, GLP-1 peptides, GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, SCFA producing enzymes, tryptophan catabolism enzymes, and/or bile salt hydrolases are secreted via Type I Hemolysin Secretion. In one embodiment, therapeutic polypeptide of interest is expressed as fusion protein with the 53 amino acids of the C terminus of alpha-hemolysin (hlyA) of E. coli CFT073.

Essential Genes and Auxotrophs

As used herein, the term “essential gene” refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, e.g., Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).

An “essential gene” may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the genetically engineered bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.

An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is a DNA synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria.

Table 28 lists depicts exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.

TABLE 28 Non-limiting Examples of Bacterial Genes Useful for Generation of an Auxotroph Amino Acid Oligonucleotide Cell Wall cysE thyA dapA glnA uraA dapB ilvD dapD leuB dapE lysA dapF serA metA glyA hisB ilvA pheA proA thrC trpC tyrA

Table 29 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli.

TABLE 29 Survival of amino acid auxotrophs in the mouse gut Gene AA Auxotroph Pre-Gavage 24 hours 48 hours argA Arginine Present Present Absent cysE Cysteine Present Present Absent glnA Glutamine Present Present Absent glyA Glycine Present Present Absent hisB Histidine Present Present Present ilvA Isoleucine Present Present Absent leuB Leucine Present Present Absent lysA Lysine Present Present Absent metA Methionine Present Present Present pheA Phenylalanine Present Present Present proA Proline Present Present Absent serA Serine Present Present Present thrC Threonine Present Present Present trpC Tryptophan Present Present Present tyrA Tyrosine Present Present Present ilvD Valine/Isoleucine/ Present Present Absent Leucine thyA Thiamine Present Absent Absent uraA Uracil Present Absent Absent flhD FlhD Present Present Present

For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria of the invention comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, 1pxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, mc, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rp1J, rp1L, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, 1spA, ispH, dapB, folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, 1pxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rp1D, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rp10, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rp1X, rp1N, rpsQ, rpmC, rp1P, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, 1pxD, fabZ, 1pxA, 1pxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, lnt, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, 1pxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, me, yceQ, fabD, fabG, acpP, tmk, holB, 1o1C, 1o1D, 1o1E, purB, ymfK, minE, mind, pth, rsA, ispE, 1o1B, hemA, prfA, prmC, kdsA, topA, ribA, fabI, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson “Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain, ”ACS Synthetic Biology (2015) DOI: 10.102¹/_(a)cssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).

In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG, and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G.

In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole.

In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L).

In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system shown in FIG. 68.

In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the genetically engineered bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). Other embodiments are described in Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-16, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (Wright et al., 2015). In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that produce the metabolic or satiety effector and/or immune modulator molecule.

Genetic Regulatory Circuits

In some embodiments, the genetically engineered bacteria comprise multi-layered genetic regulatory circuits for expressing the constructs described herein (see, e.g., U.S. Provisional Application No. 62/184,811, incorporated herein by reference in its entirety). The genetic regulatory circuits are useful to screen for mutant bacteria that produce a metabolic or satiety effector and/or immune modulator molecule or rescue an auxotroph. In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a fumarate and nitrate reductase regulator (FNR)-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule, wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the metabolic or satiety effector and/or immune modulator molecule is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf-lon protease, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a tet regulatory region (tetO); and a third gene encoding an mf-lon degradation signal linked to a tet repressor (tetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the tetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the tetR, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

Examples of repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, LacI, CscR, DeoR, DgoR, FruR, GaIR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the metabolic or satiety effector and/or immune modulator molecule. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the metabolic or satiety effector and/or immune modulator molecule from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3′ to 5′) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5′ to 3′). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the gene or gene cassette remains in the 3′ to 5′ orientation, and no functional metabolic or satiety effector and/or immune modulator molecule is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the gene or gene cassette is reverted to the 5′ to 3′ orientation, and functional metabolic or satiety effector and/or immune modulator molecule is produced.

In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule and a polymerase- and recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector and/or immune modulator molecule operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the metabolic or satiety effector and/or immune modulator molecule. The third gene encoding the T7 polymerase is inverted in orientation (3′ to 5′) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5′ to 3′). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3′ to 5′ orientation, and the metabolic or satiety effector and/or immune modulator molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5′ to 3′ orientation, and the metabolic or satiety effector and/or immune modulator molecule is expressed.

Host-Plasmid Mutual Dependency

In some embodiments, the genetically engineered bacteria of the invention also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et al., 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad-spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the anti-toxin (e.g., a wild-type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least 100 generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria of the invention.

The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxotrophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.

Synthetic gene circuits express on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a metabolic or satiety effector and/or immune modulator molecule and further comprise a toxin-antitoxin system that simultaneously produces a toxin (hok) and a short-lived antitoxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015). In some embodiments, the genetically engineered bacteria further comprise alp? from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015).

Kill Switch

In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935 and 62/263,329, incorporated herein by reference in their entireties). The kill switch is intended to actively kill genetically engineered bacteria in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.

Bacteria comprising kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a metabolic or satiety effector and/or immune modulator molecule, or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of the metabolic or satiety effector and/or immune modulator molecule, e.g., GLP-1. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of the metabolic or satiety effector and/or immune modulator molecule. Alternatively, the bacteria may be engineered to die after the bacterium has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject). Examples of such toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl P-D-1-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010).

Kill-switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g., the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.

Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in a low-oxygen environment. In some embodiments, the genetically engineered bacteria of the present disclosure comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable.

In another embodiment in which the genetically engineered bacteria of the present disclosure express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase.

In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed recombinant bacterial cell is not viable after the first essential gene is excised.

In one embodiment, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In one embodiment, the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase.

In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.

In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: Bxb1, PhiC31, TP901, Bxb1, PhiC31, TP901, HK022, HP1, R4, Intl, Int2, Int3, Int4, Int5, Int6, Intl, Int8, Int9, Int 10, Int 11, Int12, Int13, Int14, Int15, Int16, Int17, Int 18, Int19, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof.

In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill-switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present. Such kill switches are called repression-based kill switches and represent systems in which the bacterial cells are viable only in the presence of an external factor or signal, such as arabinose or other sugar. Exemplary kill switch designs in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) is shown in FIG. 69-FIG. 72. The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the AraBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxing gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arbinoase system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.

Thus, in some embodiments in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an antitoxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.

Arabinose inducible promoters are known in the art, including P_(ara), P_(araB), P_(araC), and P_(araBAD). In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the P_(araC) promoter and the P_(araBAD) promoter operate as a bidirectional promoter, with the P_(araBAD) promoter controlling expression of a heterologous gene(s) in one direction, and the P_(araC) (in close proximity to, and on the opposite strand from the P_(araBAD) promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.

In one exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P_(araBAD) promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a P_(araC) promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (P_(TetR)). In the presence of arabinose, the AraC transcription factor activates the P_(araBAD) promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the P_(araBAD) promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In one embodiment, the AraC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed.

In one embodiment of the disclosure, the genetically engineered bacterium further comprises an antitoxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the antitoxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin.

In another embodiment of the disclosure, the genetically engineered bacterium further comprises an antitoxin under the control of the P_(araBAD) promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin.

In another exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a _(ParaBAD) promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a P_(arac) promoter operably linked to a heterologous gene encoding AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the P_(araBAD) promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the P_(araBAD) promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of P_(araBAD) promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill-switch system described directly above. In some embodiments, the sequence of P_(araBAD) promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anto-toxin kill-switch system described directly above.

In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer-lived toxin killing it.

In some embodiments, the engineered bacteria of the present disclosure further comprise the gene(s) encoding the components of any of the above-described kill-switch circuits.

In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, Ibs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B17, microcin C, microcin C7-051, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin El, colicin K, colicin N, colicin U, colicin B, colicin la, colicin Ib, colicin 5, colicin10, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6, colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdID, Kis, SymR, MazE, FlmB, Sib, ptaRNA1, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBl, hipB, MccE, MccE^(cTD), MccF, Cai, ImmEl, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.

In some embodiments, the genetically engineered bacterium provided herein is an auxotroph. In one embodiment, the genetically engineered bacterium is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a ΔthyA and ΔdapA auxotroph.

In some embodiments, the genetically engineered bacterium provided herein further comprises a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

In some embodiments, the genetically engineered bacterium is an auxotroph comprising a therapeutic payload and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.

In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette for producing the metabolic or satiety effector and/or immune modulator molecule is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene or gene cassette for producing the metabolic or satiety effector and/or immune modulator molecule is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

Pharmaceutical Compositions and Formulations

Pharmaceutical compositions comprising the genetically engineered bacteria of the invention may be used to treat, manage, ameliorate, and/or prevent a metabolic disease, e.g., obesity, type 2 diabetes. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or and pharmaceutically acceptable carriers are provided.

In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria described herein that are engineered to treat, manage, ameliorate, and/or prevent a metabolic disease. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria described herein that are each engineered to treat, manage, ameliorate, and/or prevent a metabolic disease.

The pharmaceutical compositions of the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

The genetically engineered bacteria of the invention may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 10⁵ to 10¹² bacteria, e.g., approximately 10⁵ bacteria, approximately 10⁶ bacteria, approximately 10⁷ bacteria, approximately 10⁸ bacteria, approximately 10⁹ bacteria, approximately 10¹⁰ bacteria, approximately 10¹¹ bacteria, or approximately 10¹¹ bacteria. The composition may be administered once or more daily, weekly, or monthly. The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents.

The genetically engineered bacteria of the invention may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

The genetically engineered bacteria of the invention may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulo se, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers.

In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the small or large intestines. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

In some embodiments, enteric coating materials may be used, in one or more coating layers (e.g., outer, inner and/o intermediate coating layers). Enteric coated polymers remain unionised at low pH, and therefore remain insoluble. But as the pH increases in the gastrointestinal tract, the acidic functional groups are capable of ionisation, and the polymer swells or becomes soluble in the intestinal fluid.

Materials used for enteric coatings include Cellulose acetate phthalate (CAP), Poly(methacrylic acid-co-methyl methacrylate), Cellulose acetate trimellitate (CAT), Poly(vinyl acetate phthalate) (PVAP) and Hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers. Additionally, Zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch and starch derivatives, and dextrins (e.g., maltodextrin) are also used. Other known enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylo se acetate phthalate, cellulose acetate phthalate, hydroxyl propyl methyl cellulose phthalate, an ethylacrylate, and a methylmethacrylate.

Coating polymers also may comprise one or more of, phthalate derivatives, CAT, HPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylic acid ester, EudragitTM S (poly(methacrylic acid, methyl methacrylate)1:2); Eudragit L100TM S (poly(methacrylic acid, methyl methacrylate)1:1); Eudragit L3ODTM, (poly(methacrylic acid, ethyl acrylate)1:1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate)1:1) (Eudragit™ L is an anionic polymer synthesized from methacrylic acid and methacrylic acid methyl ester), polymethyl methacrylate blended with acrylic acid and acrylic ester copolymers, alginic acid, ammonia alginate, sodium, potassium, magnesium or calcium alginate, vinyl acetate copolymers, polyvinyl acetate 30D (30% dispersion in water), a neutral methacrylic ester comprising poly(dimethylaminoethylacrylate) (“Eudragit E™), a copolymer of methylmethacrylate and ethylacrylate with trimethylammonioethyl methacrylate chloride, a copolymer of methylmethacrylate and ethylacrylate, Zein, shellac, gums, or polysaccharides, or a combination thereof.

Coating layers may also include polymers which contain Hydroxypropylmethylcellulo se (HPMC), Hydroxypropylethylcellulose (HPEC), Hydroxypropylcellulo se (HPC), hydroxypropylethylcellulose (HPEC), hydroxymethylpropylcellulose (HMPC), ethylhydroxyethylcellulose (EHEC) (Ethulose), hydroxyethylmethylcellulose (HEMC), hydroxymethylethylcellulose (HMEC), propylhydroxyethylcellulose (PHEC), methylhydroxyethylcellulose (M H EC), hydrophobically modified hydroxyethylcellulose (NEXTON), carboxymethyl hydroxyethylcellulose (CMHEC), Methylcellulose, Ethylcellulose, water soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates such as ammonia alginate, sodium alginate, potassium alginate, acid phthalate of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose ester phthalates, cellulose ether phthalates, hydroxypropylcellulose phthalate (HPCP), hydroxypropylethylcellulo se phthalate (HPECP), hydroxyproplymethylcellulose phthalate (HPMCP), hydroxyproplymethylcellulose acetate succinate (HPMCAS).

Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria of the invention.

In one embodiment, the genetically engineered microorganisms of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.

In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.

In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, “flavor” is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

In certain embodiments, the genetically engineered bacteria of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

The genetically engineered bacteria of the invention may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The genetically engineered bacteria of the invention may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

In some embodiments, the invention provides pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.

Single dosage forms of the pharmaceutical composition of the invention may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

Dosage regimens may be adjusted to provide a therapeutic response. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician.

In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

The genetically engineered bacteria of the invention may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical compositions of the invention may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions of the invention is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C. and 8° C. and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD₅₀, ED₅₀, EC₅₀, and IC₅₀ may be determined, and the dose ratio between toxic and therapeutic effects (LD₅₀/ED₅₀) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

Methods of Treatment

Another aspect of the invention provides methods of treating metabolic disease, e.g., obesity, type 2 diabetes. In some embodiments, the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility. In some embodiments, the subject to be treated is a human patient.

The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria of the invention are administered orally, e.g., in a liquid suspension. In some embodiments, the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria of the invention are administered rectally, e.g., by enema. In some embodiments, the genetically engineered bacteria of the invention are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically.

In certain embodiments, the pharmaceutical composition described herein is administered to treat, manage, ameliorate, or prevent metabolic disease in a subject. In some embodiments, the method of treating or ameliorating metabolic disease allows one or more symptoms of the disease to improve by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, the symptom (e.g., obesity, insulin resistance) is measured by comparing measurements in a subject before and after administration of the pharmaceutical composition.

Before, during, and after the administration of the pharmaceutical composition in a subject, metabolic symptoms and manifestations may be measured in a biological sample, e.g., blood, serum, plasma, urine, fecal matter, peritoneal fluid, a sample collected from a tissue, such as liver, skeletal muscle, pancreas, epididymal fat, subcutaneous fat, and beige fat. The biological samples may be analyzed to measure symptoms and manifestations of metabolic disease. Useful measurements include measures of lean mass, fat mass, body weight, food intake, GLP-1 levels, endotoxin levels, insulin levels, lipid levels, HbA1c levels, short-chain fatty acid levels, triglyceride levels, and nonesterified fatty acid levels. Useful assays include, but are not limited to, insulin tolerance tests, glucose tolerance tests, pyruvate tolerance tests, assays for intestinal permeability, and assays for glycaemia upon multiple fasting and refeeding time points. In some embodiments, the methods may include administration of the compositions of the invention to reduce metabolic symptoms and manifestations to baseline levels, e.g., levels comparable to those of a healthy control, in a subject. In some embodiments, the methods may include administration of the compositions of the invention to reduce metabolic symptoms and manifestations to undetectable levels in a subject, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's levels prior to treatment.

In certain embodiments, the genetically engineered bacteria are E. coli Nissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the genetically engineered bacteria may be re-administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice is shown in FIG. 84 and FIG. 85. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.

The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents, e.g., insulin. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

Treatment In Vivo

The genetically engineered bacteria of the invention may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a metabolic disease may be used (see, e.g., Mizoguchi 2012). In some embodiments, the animal is a C57BL/6J mouse that is fed a high fat diet in order to induce obesity and T2DM-related symptoms such as hyperinsulinemia and hyperglycemia. In alternate embodiments, an animal harboring a genetic deficiency that causes a metabolic disease, e.g., a B6.BKS(D)-Lepr^(db/db) mouse, is used.

The genetically engineered bacteria of the invention are administered to the mice before, during, or after the onset of obesity and disease. Body weight, food intake, and blood plasma (e.g., triglyceride levels, insulin tolerance tests, glucose tolerance tests, pyruvate tolerance tests) may be assayed to determine the severity and amelioration of disease. Metabolism and physical activity may be measured in metabolic cages. Animals may be sacrificed to assay metabolic tissues such as liver, skeletal muscle, epididymal fat, subcutaneous fat, brown fat, pancreas, and brain, are collected for analysis of histology and gene expression.

TABLE 30 Summary of rodent models of type 2 diabetes Induction mechanism Model Main features Possible uses Obese models Lep^(ob/ob) mice Obesity-induced Treatments to improve (monogenic) hyperglycaemia insulin resistance Lepr^(db/db) mice Treatments to improve ZDF Rats beta cell function Obese models KK mice Obesity-induced Treatments to improve (polygenic) hyperglycaemia insulin resistance OLETF rat Treatments to improve beta cell function NZO mice Some models show TallyHo/Jng mice diabetic complications NoncNZO10/LtJ mice Induced obesity High fat feeding (mice Obesity-induced Treatments to improve or rats) hyperglycaemia insulin resistance Desert gerbil Treatments to improve beta cell function Nile grass rat Treatments to prevent diet-induced obesity Non-obese GK rat Hyperglycaemia Treatments to improve models induced by beta cell function insufficient beta Treatments to improve cell function/mass beta cell survival Genetically hIAPP mice Amyloid Treatments to prevent induced models deposition in islets amyloid deposition of beta cell Treatments to improve dysfunction beta cell survival AKITA mice Beta cell Treatments to prevent ER destruction due to stress ER stress. Treatments to improve beta cell survival

As described in Aileen JF King, The use of animal models in diabetes research, Br J Pharmacol. 2012 June; 166(3): 877-894.

The engineered bacteria may be evaluated in vivo, e.g., in an animal model for NASH. Any suitable animal model of a disease associated with Non-Alcoholic Fatty Liver Disease/Non-Alcoholic Steatohepatitis (NAFLD/NASH) may be used. For example, the effects of liver steatosis and hepatic inflammation in an in vivo mouse model have been described (Jun Jin, et al., Brit. J. Nutrition, 114:145-1755 (2015)). To briefly summarize, female C57BL/6J mice can be fasted and fed either a standard liquid diet of carbohydrates, fat, and protein; or a liquid Western style diet (WSD) fortified with fructose, fat, cholesterol, and a sodium butyrate supplement for six weeks. Butyrate is a short chain fatty acid naturally produced by intestinal bacteria effective in maintaining intestinal homoeostasis. Body weight and plasma samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and the liver and intestine can be removed and assayed.

An in vivo rat model of choline deficient/L-amino acid defined (CDAA) diet has also been described (Endo, et al., PLoS One, 8(5):e63388 (2013)). In this model, rats are fed the CDAA diet for eight weeks and then treated with a strain of Clostridium butyricum (MIYAIRI 588) two weeks after. The diet induces NAFLD/NASH symptoms such as liver steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocarcinogenesis. The rats are killed at 8, 16, and 50 weeks after completion of the diet regiments, and liver tissues removed and assayed.

Other models are known in the art, including a Lepob/Lepob and C57BL6 (B6) mouse model used to study the effects of high fat diet and GLP-1 administration within the NASH setting. See, for example, Trevaskis et al., Am. J. Physiology-Gastrointestinal and Liver Physiology, 302(8):G762-G772, 2012, and Takahashi et al., World J. Gastroenterol., 18(19):2300-2308, 2012, the entire contents of each of which are expressly incorporated herein by reference.

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EXAMPLES

The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.

Example 1 Construction of Vectors for Producing Propionate

To facilitate inducible production of propionate in Escherichia coli Nissle, a propionate gene cassette comprising the genes encoding the enzymes of the acrylate pathway, i.e., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The genes are codon-optimized for E. coli codon usage using Integrated DNA Technologies online codon optimization tool (https://www.idtdna.com/CodonOpt). A second clone is generated as described above using a propionate gene cassette comprising the genes encoding the enzymes of the pyruvate pathway, i.e., thrA^(fr), thrB, thrC, ilvA^(fbr), aceE, aceF, and lpd; NCBI; Tseng et al., 2012). A third clone is generated as described above that comprises thrA^(fbr), thrB, thrC, ilvA^(fbr), aceE, aceF, lpd, and E. coli tesB. Each propionate gene cassette is expressed under the control of each of the following regulatory regions: a FNR-inducible regulatory region selected from the sequences listed in Table 21, a tetracycline-inducible promoter, and an arabinose-inducible promoter. In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of propionate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site. Each gene cassette and regulatory region construct is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome.

The propionate construct is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., FIG. 57). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. At the site of insertion, DNA primers that are homologous to the site of insertion and to the propionate construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below. The resulting E. coli Nissle bacteria are genetically engineered to express a propionate biosynthesis cassette and produce propionate.

Example 2 Lambda Red Recombination

Lambda red recombination is used to make chromosomal modifications, e.g., to express a propionate biosynthesis cassette in E. coli Nissle. Lambda red is a procedure using recombination enzymes from a bacteriophage lambda to insert a piece of custom DNA into the chromosome of E. coli . A pKD46 plasmid is transformed into the E. coli Nissle host strain. E. coli Nissle cells are grown overnight in LB media. The overnight culture is diluted 1:100 in 5 mL of LB media and grown until it reaches an OD₆₀₀ of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 1 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 1 ng of pKD46 plasmid DNA is added to the E. coli cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C. for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C.

DNA sequences comprising the desired propionate biosynthesis genes shown above were ordered from a gene synthesis company. The lambda enzymes are used to insert this construct into the genome of E. coli Nissle through homologous recombination. The construct is inserted into a specific site in the genome of E. coli Nissle based on its DNA sequence. In some embodiments, the construct is in the E. coli Nissle genome at the malP/T site (FIG. 57). To insert the construct into a specific site, the homologous DNA sequence flanking the construct is identified, and includes approximately 50 bases on either side of the sequence. The homologous sequences are ordered as part of the synthesized gene. Alternatively, the homologous sequences may be added by PCR. The construct includes an antibiotic resistance marker that may be removed by recombination. The resulting construct comprises approximately 50 bases of homology upstream, a kanamycin resistance marker that can be removed by recombination, the propionate biosynthesis genes, and approximately 50 bases of homology downstream.

Example 3 Transforming E. coli

Each of the constructs above is transformed into E. coli Nissle comprising pKD46. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture is diluted 1:100 in 5 mL of LB media containing ampicillin and grown until it reaches an OD₆₀₀ of 0.1. 0.05 mL of 100× L-arabinose stock solution is added to induce pKD46 lambda red expression. The culture is grown until it reaches an OD₆₀₀ of 0.4-0.6. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 1 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C. water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 0.5 μg of the construct is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C. for 1 hr. The cells are spread out on an LB plate containing kanamycin and incubated overnight.

In alternate embodiments, the propionate cassette may be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, Mass.). Organization of the constructs and nucleotide sequences are shown in FIGS. 1-5. To create a vector capable of integrating the synthesized propionate cassette construct into the chromosome, Gibson assembly was first used to add 1000 bp sequences of DNA homologous to the Nissle lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the lacZ locus in the Nissle genome. Gibson assembly was used to clone the fragment between these arms. PCR was used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the propionate cassette between them. This PCR fragment was used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells were grown out for 2 hours before plating on chloramphenicol at 20 ug/mL at 37 degrees C. Growth at 37 degrees C. also cures the pKD46 plasmid. Transformants containing cassette were chloramphenicol resistant and lac-minus (lac−).

Example 4 Verifying Mutants

The presence of the propionate gene cassette is verified by colony PCR. Colonies are picked with a pipette tip and resuspended in 20 pi of cold ddH₂O by pipetting up and down. 3 μl of the suspension is pipetted onto an index plate with appropriate antibiotic for use later. The index plate is grown at 37° C. overnight. A PCR master mix is made using 5 μl of 10× PCR buffer, 0.6 μl of 10 mM dNTPs, 0.4 μl of 50 mM Mg₂SO₄, 6.0 μl of 10× enhancer, and 3.0 μl of ddH₂O (15 μl of master mix per PCR reaction). A 10 μM primer mix is made by mixing 2 μL of primers unique to the propionate construct (100 μM stock) into 16 μL of ddH₂O. For each 20 μl reaction, 15 μL of the PCR master mix, 2.0 μL of the colony suspension (template), 2.0 μL of the primer mix, and 1.0 μL of Pfx Platinum DNA Pol are mixed in a PCR tube. The PCR thermocycler is programmed as follows, with steps 2-4 repeating 34 times: 1) 94° C. at 5:00 min., 2) 94° C. at 0:15 min., 3) 55° C. at 0:30 min., 4) 68° C. at 2:00 min., 5) 68° C. at 7:00 min., and then cooled to 4° C. The PCR products are analyzed by gel electrophoresis using 10 μL of each amplicon and 2.5 μL 5× dye. The PCR product only forms if the heterologous sequence has been inserted.

Example 5 Generation of ΔThyA

An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thyA, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine.

A thyA::cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a 100 um concentration are found in Table 31.

TABLE 31 Primer Sequences SEQ ID Name Sequence Description NO SR36 tagaactgatgcaaaaagtgctcgacgaaggcacacagaTGTGTAG Round 1: binds SEQ ID GCTGGAGCTGCTTC on pKD3 NO: 215 SR38 gtttcgtaattagatagccaccggcgctttaatgcccggaCATATGAA Round 1: binds SEQ ID TATCCTCCTTAG on pKD3 NO: 216 SR33 caacacgtttcctgaggaaccatgaaacagtatttagaactgatgcaaaaag Round 2: binds SEQ ID to round 1 PCR NO: 217 product SR34 cgcacactggcgtcggctctggcaggatgtttcgtaattagatagc Round 2: binds SEQ ID to round 1 PCR NO: 218 product SR43 atatcgtcgcagcccacagcaacacgtttcctgagg Round 3: binds SEQ ID to round 2 PCR NO: 219 product SR44 aagaatttaacggagggcaaaaaaaaccgacgcacactggcgtcggc Round 3: binds SEQ ID to round 2 PCR NO: 220 product

For the first PCR round, 4×50 ul PCR reactions containing ing pKD3 as template, 25 ul 2×phusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6 ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions:

stepl: 98 c for 30 s

step2: 98 c for 10 s

step3: 55 c for 15 s

step4: 72 c for 20 s

repeat step 2-4 for 30 cycles

step5: 72 c for 5 min

Subsequently, 5 ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer's instructions and eluted in 30 ul nuclease free water.

For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4×50 ul PCR reactions as described above except with 0.2 ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30 ul as described above.

For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4×50 ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, lml SOC medium containing 3 mM thymidine was added, and cells were allowed to recover at 37 C for 2 h with shaking. Cells were then pelleted at 10,000×g for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100 ul LB containing 3 mM thymidine and spread on LB agar plates containing 3 mM thy and 20 ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. +cam 20 ug/ml + or − thy 3 mM. (thyA auxotrophs will only grow in media supplemented with thy 3 mM).

Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37° C. until OD600=0.4-0.6. 1mL of cells were washed as follows: cells were pelleted at 16,000×g for 1 minute. The supernatant was discarded and the pellet was resuspended in 1 mL ice-cold 10% glycerol. This wash step was repeated 3× times. The final pellet was resuspended in 70 ul ice-cold 10% glycerol. Next, cells were electroporated with ing pCP20 plasmid DNA, and 1 mL SOC supplemented with 3 mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30° C. for lhours. Cells were then pelleted at 10,000×g for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100 ul LB containing 3 mM thymidine and spread on LB agar plates containing 3 mM thy and 100ug/mlcarbenicillin and grown at 30° C. for 16-24 hours. Next, transformants were colony purified non-selectively (no antibiotics) at 42° C.

To test the colony-purified transformants, a colony was picked from the 42° C. plate with a pipette tip and resuspended in 10 μL LB. 3 μl, of the cell suspension was pipetted onto a set of 3 plates: Cam, (37° C.; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30° C., tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37° C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37° C.

Example 6 Production of Propionate in Genetically Engineered E. coli

Production of propionate is assessed in E. coli Nissle strains containing the propionate cassettes described above. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the propionate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h, and the inducible constructs are induced as follows: (1) bacteria comprising a propionate gene cassette driven by a FNR-inducible promoter are induced in LB at 37C for up to 4 hours in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2, and 20 mM nitrate) at 37° C.; (2) bacteria comprising a propionate gene cassette driven by a tetracycline-inducible promoter are induced with anhydrotetracycline (100 ng/mL); (3) bacteria comprising a propionate gene cassette driven by a arabinose-inducible promoter are induced with 1% arabinose in media lacking glucose. One mL culture aliquots are prepared in 1.5 mL capped tubes and FNR-inducible constructs are incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for propionate concentration by LC-MS to confirm that propionate production in these recombinant strains can be achieved in a low-oxygen environment.

Example 7 Efficacy of Propionate-Expressing Bacteria in a Mouse Model of Obesity and Type 2 Diabetes Mellitus (T2DM)

For in vivo studies to assess the efficacy of the genetically engineered bacteria in an animal model of obesity and type 2 diabetes, C57BL/6J mice are fed a high fat diet (60 kcal % fat, Research Diets Inc.) starting from 4-5 weeks of age for 8 weeks or until body weight is at least 45 g in order to induce obesity and T2DM-related symptoms such as hyperinsulinemia and hyperglycemia, e.g., glycaemia above 160 mg/dL and plasma insulin above 4000 pg/mL. Alternatively, B6.BKS(D)-Lepr^(db/db) mice (Lepr^(db/db)) are obtained from The Jackson Laboratory; these mice typically become obese and display T2DM-related symptoms beginning at 10 weeks of age.

Bacteria harboring the propionate gene cassette described above are grown overnight in LB. Bacteria are then diluted 1:100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. To analyze the efficacy of the bacteria in vivo, bacteria are resuspended in phosphate buffered saline (PBS) and 100 microliters is administered by oral gavage to mice daily for 8 weeks. Alternatively, the bacteria can be supplemented in the drinking water (5×10⁹ CFU bacteria/mL).

Body weight and food intake are measured weekly before, during, and after the administration of the bacteria. In addition, mice are subjected to insulin tolerance tests (ITT), glucose tolerance tests (GTT) and pyruvate tolerance tests (PTT) to determine the severity of T2DM during treatment, e.g., amelioration of insulin resistance. For ITT, mice are fasted overnight and injected with insulin (1 U/kg, diluted in PBS). Blood glucose levels are measured prior to the injection and at 20, 40, 60, and 90 min. post injection via tail bleeding. For GTT, mice are fasted overnight and injected with glucose solution (1 g/kg, dissolved in PBS); blood glucose levels are measured as described above in order to determine changes. For PTT, mice are fasted overnight and injected with sodium pyruvate solution (lg/kg, dissolved in PBS); blood glucose levels are measured as described above. Whole-body metabolic functions are analyzed by placing the mice in a Comprehensive Lab Animal Monitoring System (CLAMS), which monitors physical activity, food intake, metabolic rate (as a function of O₂ consumption and CO₂ production). Mice are sacrificed and metabolic tissues such as liver, skeletal muscle, epididymal fat, subcutaneous fat, brown fat, pancreas, and brain, are collected for analysis of histology, e.g., Oil Red O staining of the liver, and gene expression.

Example 8 Nissle Residence

Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non-limiting example using a streptomycin-resistant strain of E. coli Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention.

C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation (i.e., day 0), streptomycin-resistant Nissle (SYN-103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 32. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37° C. overnight, and colonies were counted.

TABLE 32 CFU administered via oral gavage CFU administered via oral gavage Strain Day 1 Day 2 Day 3 SYN-103 1.30E+08 8.50E+08 1.90E+09

On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs. 1-6; Table 14). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37° C. overnight, and colonies were counted.

Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 μg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 33.

FIG. 84 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

TABLE 33 Nissle residence in vivo ID Day 2 Day 3 Day 4 Day 5 1 2.40E+05 6.50E+03 6.00E+04 2.00E+03 2 1.00E+05 1.00E+04 3.30E+04 3.00E+03 3 6.00E+04 1.70E+04 6.30E+04 2.00E+02 4 3.00E+04 1.50E+04 1.10E+05 3.00E+02 5 1.00E+04 3.00E+05 1.50E+04 6 1.00E+06 4.00E+05 2.30E+04 Avg 1.08E+05 1.76E+05 1.61E+05 7.25E+03 ID Day 6 Day 7 Day 8 Day 9 Day 10 1 9.10E+03 1.70E+03 4.30E+03 6.40E+03 2.77E+03 2 6.00E+03 7.00E+02 6.00E+02 0.00E+00 0.00E+00 3 1.00E+02 2.00E+02 0.00E+00 0.00E+00 0.00E+00 4 1.50E+03 1.00E+02 0.00E+00 0.00E+00 5 3.10E+04 3.60E+03 0.00E+00 0.00E+00 6 1.50E+03 1.40E+03 4.20E+03 1.00E+02 0.00E+00 Avg 8.20E+03 1.28E+03 2.28E+03 1.08E+03 4.62E+02

Example 9 Intestinal Residence and Survival of Bacterial Strains In Vivo

Localization and intestinal residence time of streptomycin resistant Nissle, FIG. 85) was determined. Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon.

Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 10¹⁰ CFU/mL. Mice (C57BL6/J, 10-12 weeks old) were gavaged with 100 μL of bacteria (approximately 10⁹ CFU). Drinking water for the mice was changed to contain 0.1 mg/mL anhydrotetracycline (ATC) and 5% sucrose for palatability. At each timepoint (1, 4, 8, 12, 24, and 30 hours post-gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating.

In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37° C. overnight, and colonies were counted. The amount of bacteria and residence time in each compartment is shown in FIG. 85.

Example 10 Construction of Vectors for Overproducing Butyrate

In addition to the ammonia conversion circuit, GABA transport circuit, GABA metabolic circuit, and/or manganese transport circuit described above, the E. coli Nissle bacteria further comprise one or more circuits for producing a gut barrier enhancer molecule.

To facilitate inducible production of butyrate in E. coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiA1, hbd, crt2, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The butyrate gene cassette is placed under the control of a FNR regulatory region selected from (SEQ ID NOs: 177-188) (Table 18 or Table 19) In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

In certain constructs, the butyrate gene cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Tables 34 and 35). In certain constructs, the butyrate gene cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Tables 14-17). In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter.

TABLE 34 pLogic031-nsrR-norB-butyrate construct (SEQ ID NO: 221) Nucleotide sequences of pLogic031-nsrR-norB-butyrate Description construct (SEQ ID NO: 221) Nucleic acid ttatta tcgcaccgcaatcgggattttcgattcataaagcagg sequence of an tcgtaggtcggcttgttgagcaggtcttgcagcgtgaaaccgt exemplary RNS- ccagatacgtgaaaaacgacttcattgcaccgccgagtatgcc regulated cgtcagccggcaggacggcgtaatcaggcattcgttgttcggg construct cccatacactcgaccagctgcatcggttcgaggtggcggacga comprising a gene ccgcgccgatattgatgcgttcgggcggcgcggccagcctcag encoding nsrR, a cccgccgcctttcccgcgtacgctgtgcaagaacccgcctttg regulatory region accagcgcggtaaccactttcatcaaatggcttttggaaatgc of norB, and a cgtaggtcgaggcgatggtggcgatattgaccagcgcgtcgtc butyrogenic gene gttgacggcggtgtagatgaggacgcgcagcccgtagtcggta cassette tgttgggtcagatacat acaacctccttagtacatgcaaaatt (pLogic031-nsrR- atttctagagcaacatacgagccggaagcataaagtgtaaagc norB-butyrate ctggggtgcctaatgagttgagttgaggaattataacaggaag construct; SEQ. ID aaatattcctcatacgcttgtaattcctctatggttgttgaca NO: 79). The

sequence

encoding NsrR is gatatacatatggatttaaattctaaaaaatatcagatgctta underlined and aagagctatatgtaagcttcgctgaaaatgaagttaaaccttt bolded , and the agcaacagaacttgatgaagaagaaagatttccttatgaaaca NsrR binding site, gtggaaaaaatggcaaaagcaggaatgatgggtataccatatc i.e., a regulatory caaaagaatatggtggagaaggtggagacactgtaggatatat region of norB is aatggcagttgaagaattgtctagagtttgtggtactacagga

gttatattatcagctcatacatctcttggctcatggcctatat atcaatatggtaatgaagaacaaaaacaaaaattcttaagacc actagcaagtggagaaaaattaggagcatttggtcttactgag cctaatgctggtacagatgcgtctggccaacaaacaactgctg ttttagacggggatgaatacatacttaatggctcaaaaatatt tataacaaacgcaatagctggtgacatatatgtagtaatggca atgactgataaatctaaggggaacaaaggaatatcagcattta tagttgaaaaaggaactcctgggtttagctttggagttaaaga aaagaaaatgggtataagaggttcagctacgagtgaattaata tttgaggattgcagaatacctaaagaaaatttacttggaaaag aaggtcaaggatttaagatagcaatgtctactcttgatggtgg tagaattggtatagctgcacaagctttaggtttagcacaaggt gctcttgatgaaactgttaaatatgtaaaagaaagagtacaat ttggtagaccattatcaaaattccaaaatacacaattccaatt agctgatatggaagttaaggtacaagcggctagacaccttgta tatcaagcagctataaataaagacttaggaaaaccttatggag tagaagcagcaatggcaaaattatttgcagctgaaacagctat ggaagttactacaaaagctgtacaacttcatggaggatatgga tacactcgtgactatccagtagaaagaatgatgagagatgcta agataactgaaatatatgaaggaactagtgaagttcaaagaat ggttatttcaggaaaactattaaaatagtaagaaggagatata catatggaggaaggatttatgaatatagtcgtttgtataaaac aagttccagatacaacagaagttaaactagatcctaatacagg tactttaattagagatggagtaccaagtataataaaccctgat gataaagcaggtttagaagaagctataaaattaaaagaagaaa tgggtgctcatgtaactgttataacaatgggacctcctcaagc agatatggctttaaaagaagctttagcaatgggtgcagataga ggtatattattaacagatagagcatttgcgggtgctgatactt gggcaacttcatcagcattagcaggagcattaaaaaatataga ttttgatattataatagctggaagacaggcgatagatggagat actgcacaagttggacctcaaatagctgaacatttaaatcttc catcaataacatatgctgaagaaataaaaactgaaggtgaata tgtattagtaaaaagacaatttgaagattgttgccatgactta aaagttaaaatgccatgccttataacaactcttaaagatatga acacaccaagatacatgaaagttggaagaatatatgatgcttt cgaaaatgatgtagtagaaacatggactgtaaaagatatagaa gttgacccttctaatttaggtcttaaaggttctccaactagtg tatttaaatcatttacaaaatcagttaaaccagctggtacaat atacaatgaagatgcgaaaacatcagctggaattatcatagat aaattaaaagagaagtatatcatataataagaaggagatatac atatgggtaacgttttagtagtaatagaacaaagagaaaatgt aattcaaactgtttctttagaattactaggaaaggctacagaa atagcaaaagattatgatacaaaagtttctgcattacttttag gtagtaaggtagaaggtttaatagatacattagcacactatgg tgcagatgaggtaatagtagtagatgatgaagctttagcagtg tatacaactgaaccatatacaaaagcagcttatgaagcaataa aagcagctgaccctatagttgtattatttggtgcaacttcaat aggtagagatttagcgcctagagtttctgctagaatacataca ggtcttactgctgactgtacaggtcttgcagtagctgaagata caaaattattattaatgacaagacctgcctttggtggaaatat aatggcaacaatagtttgtaaagatttcagacctcaaatgtct acagttagaccaggggttatgaagaaaaatgaacctgatgaaa ctaaagaagctgtaattaaccgtttcaaggtagaatttaatga tgctgataaattagttcaagttgtacaagtaataaaagaagct aaaaaacaagttaaaatagaagatgctaagatattagtttctg ctggacgtggaatgggtggaaaagaaaacttagacatacttta tgaattagctgaaattataggtggagaagtttctggttctcgt gccactatagatgcaggttggttagataaagcaagacaagttg gtcaaactggtaaaactgtaagaccagacctttatatagcatg tggtatatctggagcaatacaacatatagctggtatggaagat gctgagtttatagttgctataaataaaaatccagaagctccaa tatttaaatatgctgatgttggtatagttggagatgttcataa agtgcttccagaacttatcagtcagttaagtgttgcaaaagaa aaaggtgaagttttagctaactaataagaaggagatatacata tgagagaagtagtaattgccagtgcagctagaacagcagtagg aagttttggaggagcatttaaatcagtttcagcggtagagtta ggggtaacagcagctaaagaagctataaaaagagctaacataa ctccagatatgatagatgaatctcttttagggggagtacttac agcaggtcttggacaaaatatagcaagacaaatagcattagga gcaggaataccagtagaaaaaccagctatgactataaatatag tttgtggttctggattaagatctgtttcaatggcatctcaact tatagcattaggtgatgctgatataatgttagttggtggagct gaaaacatgagtatgtctccttatttagtaccaagtgcgagat atggtgcaagaatgggtgatgctgcttttgttgattcaatgat aaaagatggattatcagacatatttaataactatcacatgggt attactgctgaaaacatagcagagcaatggaatataactagag aagaacaagatgaattagctcttgcaagtcaaaataaagctga aaaagctcaagctgaaggaaaatttgatgaagaaatagttcct gttgttataaaaggaagaaaaggtgacactgtagtagataaag atgaatatattaagcctggcactacaatggagaaacttgctaa gttaagacctgcatttaaaaaagatggaacagttactgctggt aatgcatcaggaataaatgatggtgctgctatgttagtagtaa tggctaaagaaaaagctgaagaactaggaatagagcctcttgc aactatagtttcttatggaacagctggtgttgaccctaaaata atgggatatggaccagttccagcaactaaaaaagctttagaag ctgctaatatgactattgaagatatagatttagttgaagctaa tgaggcatttgctgcccaatctgtagctgtaataagagactta aatatagatatgaataaagttaatgttaatggtggagcaatag ctataggacatccaataggatgctcaggagcaagaatacttac tacacttttatatgaaatgaagagaagagatgctaaaactggt cttgctacactttgtataggcggtggaatgggaactactttaa tagttaagagatagtaagaaggagatatacatatgaaattagc tgtaataggtagtggaactatgggaagtggtattgtacaaact tttgcaagttgtggacatgatgtatgtttaaagagtagaactc aaggtgctatagataaatgtttagctttattagataaaaattt aactaagttagttactaagggaaaaatggatgaagctacaaaa gcagaaatattaagtcatgttagttcaactactaattatgaag atttaaaagatatggatttaataatagaagcatctgtagaaga catgaatataaagaaagatgttttcaagttactagatgaatta tgtaaagaagatactatcttggcaacaaatacttcatcattat ctataacagaaatagcttcttctactaagcgcccagataaagt tataggaatgcatttctttaatccagttcctatgatgaaatta gttgaagttataagtggtcagttaacatcaaaagttacttttg atacagtatttgaattatctaagagtatcaataaagtaccagt agatgtatctgaatctcctggatttgtagtaaatagaatactt atacctatgataaatgaagctgttggtatatatgcagatggtg ttgcaagtaaagaagaaatagatgaagctatgaaattaggagc aaaccatccaatgggaccactagcattaggtgatttaatcgga ttagatgttgttttagctataatgaacgttttatatactgaat ttggagatactaaatatagacctcatccacttttagctaaaat ggttagagctaatcaattaggaagaaaaactaagataggattc tatgattataataaataataagaaggagatatacatatgagta caagtgatgttaaagtttatgagaatgtagctgttgaagtaga tggaaatatatgtacagtgaaaatgaatagacctaaagccctt aatgcaataaattcaaagactttagaagaactttatgaagtat ttgtagatattaataatgatgaaactattgatgttgtaatatt gacaggggaaggaaaggcatttgtagctggagcagatattgca tacatgaaagatttagatgctgtagctgctaaagattttagta tcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa agtagtgatagctgctgtaaacggatttgctttaggtggagga tgtgaacttgcaatggcatgtgatataagaattgcatctgcta aagctaaatttggtcagccagaagtaactcttggaataactcc aggatatggaggaactcaaaggcttacaagattggttggaatg gcaaaagcaaaagaattaatctttacaggtcaagttataaaag ctgatgaagctgaaaaaatagggctagtaaatagagtcgttga gccagacattttaatagaagaagttgagaaattagctaagata atagctaaaaatgctcagcttgcagttagatactctaaagaag caatacaacttggtgctcaaactgatataaatactggaataga tatagaatctaatttatttggtctttgtttttcaactaaagac caaaaagaaggaatgtcagctttcgttgaaaagagagaagcta actttataaaagggtaataagaaggagatatacatatgagaag ttttgaagaagtaattaagtttgcaaaagaaagaggacctaaa actatatcagtagcatgttgccaagataaagaagttttaatgg cagttgaaatggctagaaaagaaaaaatagcaaatgccatttt agtaggagatatagaaaagactaaagaaattgcaaaaagcata gacatggatatcgaaaattatgaactgatagatataaaagatt tagcagaagcatctctaaaatctgttgaattagtttcacaagg aaaagccgacatggtaatgaaaggcttagtagacacatcaata atactaaaagcagttttaaataaagaagtaggtcttagaactg gaaatgtattaagtcacgtagcagtatttgatgtagagggata tgatagattatttttcgtaactgacgcagctatgaacttagct cctgatacaaatactaaaaagcaaatcatagaaaatgcttgca cagtagcacattcattagatataagtgaaccaaaagttgctgc aatatgcgcaaaagaaaaagtaaatccaaaaatgaaagataca gttgaagctaaagaactagaagaaatgtatgaaagaggagaaa tcaaaggttgtatggttggtgggccttttgcaattgataatgc agtatctttagaagcagctaaacataaaggtataaatcatcct gtagcaggacgagctgatatattattagccccagatattgaag gtggtaacatattatataaagctttggtattcttctcaaaatc aaaaaatgcaggagttatagttggggctaaagcaccaataata ttaacttctagagcagacagtgaagaaactaaactaaactcaa tagctttaggtgttttaatggcagcaaaggcataataagaagg agatatacatatgagcaaaatatttaaaatcttaacaataaat cctggttcgacatcaactaaaatagctgtatttgataatgagg atttagtatttgaaaaaactttaagacattcttcagaagaaat aggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaa caagtaatagaagaagctctaaaagaaggtggagtaaaaacat ctgaattagatgctgtagtaggtagaggaggacttcttaaacc tataaaaggtggtacttattcagtaagtgctgctatgattgaa gatttaaaagtgggagttttaggagaacacgcttcaaacctag gtggaataatagcaaaacaaataggtgaagaagtaaatgttcc ttcatacatagtagaccctgttgttgtagatgaattagaagat gttgctagaatttctggtatgcctgaaataagtagagcaagtg tagtacatgctttaaatcaaaaggcaatagcaagaagatatgc tagagaaataaacaagaaatatgaagatataaatcttatagtt gcacacatgggtggaggagtttctgttggagctcataaaaatg gtaaaatagtagatgttgcaaacgcattagatggagaaggacc tttctctccagaaagaagtggtggactaccagtaggtgcatta gtaaaaatgtgctttagtggaaaatatactcaagatgaaatta aaaagaaaataaaaggtaatggcggactagttgcatacttaaa cactaatgatgctagagaagttgaagaaagaattgaagctggt gatgaaaaagctaaattagtatatgaagctatggcatatcaaa tctctaaagaaataggagctagtgctgcagttcttaagggaga tgtaaaagcaatattattaactggtggaatcgcatattcaaaa atgtttacagaaatgattgcagatagagttaaatttatagcag atgtaaaagtttatccaggtgaagatgaaatgattgcattagc tcaaggtggacttagagttttaactggtgaagaagaggctcaa gtttatgataactaataa

TABLE 35 Nucleotide sequences of pLogic046-nsrR-norB-butyrate construct Nucleotide sequences of pLogic046-nsrR-norB-butyrate construct Description (SEQ ID NO: 222) Nucleic acid ttatta tcgcaccgcaatcgggattttcgattcataaagcaggtc sequence of an gtaggtcggcttgttgagcaggtcttgcagcgtgaaaccgtccag exemplary RNS- atacgtgaaaaacgacttcattgcaccgccgagtatgcccgtcag regulated ccggcaggacggcgtaatcaggcattcgttgttcgggcccataca construct ctcgaccagctgcatcggttcgaggtggcggacgaccgcgccgat comprising a attgatgcgttcgggcggcgcggccagcctcagcccgccgccttt gene encoding cccgcgtacgctgtgcaagaacccgcctttgaccagcgcggtaac nsrR, a cactttcatcaaatggcttttggaaatgccgtaggtcgaggcgat regulatory ggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagat region of norB, gaggacgcgcagcccgtagtcggtatgttgggtcagatacat aca and a acctccttagtacatgcaaaattatttctagagcaacatacgagc butyrogenic cggaagcataaagtgtaaagcctggggtgcctaatgagttgagtt gene cassette gaggaattataacaggaagaaatattcctcatacgcttgtaattc (pLogic046-

nsrR-norB-

butyrate actttaagaaggagatatacatatgatcgtaaaacctatggtacg construct; SEQ. caacaatatctgcctgaacgcccatcctcagggctgcaagaaggg ID NO: 80). agtggaagatcagattgaatataccaagaaacgcattaccgcaga agtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttgg ctgctcaaatggttacggcctggcgagccgcattactgctgcgtt cggatacggggctgcgaccatcggcgtgtcctttgaaaaagcggg ttcagaaaccaaatatggtacaccgggatggtacaataatttggc atttgatgaagcggcaaaacgcgagggtctttatagcgtgacgat cgacggcgatgcgttttcagacgagatcaaggcccaggtaattga ggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacag cttggccagcccagtacgtactgatcctgatacaggtatcatgca caaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaac agtagatccgtttactggcgagctgaaggaaatctccgcggaacc agcaaatgacgaggaagcagccgccactgttaaagttatgggggg tgaagattgggaacgttggattaagcagctgtcgaaggaaggcct cttagaagaaggctgtattaccttggcctatagttatattggccc tgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggc caaagaacacctggaggccacagcacaccgtctcaacaaagagaa cccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaac ccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccag cttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtat tgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaaga tggtacaattccagttgatgaggaaaatcgcattcgcattgatga ttgggagttagaagaagacgtccagaaagcggtatccgcgttgat ggagaaagtcacgggtgaaaacgcagaatctctcactgacttagc ggggtaccgccatgatttcttagctagtaacggctttgatgtaga aggtattaattatgaagcggaagttgaacgcttcgaccgtatctg ataagaaggagatatacatatgagagaagtagtaattgccagtgc agctagaacagcagtaggaagttttggaggagcatttaaatcagt ttcagcggtagagttaggggtaacagcagctaaagaagctataaa aagagctaacataactccagatatgatagatgaatctcttttagg gggagtacttacagcaggtcttggacaaaatatagcaagacaaat agcattaggagcaggaataccagtagaaaaaccagctatgactat aaatatagtttgtggttctggattaagatctgtttcaatggcatc tcaacttatagcattaggtgatgctgatataatgttagttggtgg agctgaaaacatgagtatgtctccttatttagtaccaagtgcgag atatggtgcaagaatgggtgatgctgcttttgttgattcaatgat aaaagatggattatcagacatatttaataactatcacatgggtat tactgctgaaaacatagcagagcaatggaatataactagagaaga acaagatgaattagctcttgcaagtcaaaataaagctgaaaaagc tcaagctgaaggaaaatttgatgaagaaatagttcctgttgttat aaaaggaagaaaaggtgacactgtagtagataaagatgaatatat taagcctggcactacaatggagaaacttgctaagttaagacctgc atttaaaaaagatggaacagttactgctggtaatgcatcaggaat aaatgatggtgctgctatgttagtagtaatggctaaagaaaaagc tgaagaactaggaatagagcctcttgcaactatagtttcttatgg aacagctggtgttgaccctaaaataatgggatatggaccagttcc agcaactaaaaaagctttagaagctgctaatatgactattgaaga tatagatttagttgaagctaatgaggcatttgctgcccaatctgt agctgtaataagagacttaaatatagatatgaataaagttaatgt taatggtggagcaatagctataggacatccaataggatgctcagg agcaagaatacttactacacttttatatgaaatgaagagaagaga tgctaaaactggtcttgctacactttgtataggcggtggaatggg aactactttaatagttaagagatagtaagaaggagatatacatat gaaattagctgtaataggtagtggaactatgggaagtggtattgt acaaacttttgcaagttgtggacatgatgtatgtttaaagagtag aactcaaggtgctatagataaatgtttagctttattagataaaaa tttaactaagttagttactaagggaaaaatggatgaagctacaaa agcagaaatattaagtcatgttagttcaactactaattatgaaga tttaaaagatatggatttaataatagaagcatctgtagaagacat gaatataaagaaagatgttttcaagttactagatgaattatgtaa agaagatactatcttggcaacaaatacttcatcattatctataac agaaatagcttcttctactaagcgcccagataaagttataggaat gcatttctttaatccagttcctatgatgaaattagttgaagttat aagtggtcagttaacatcaaaagttacttttgatacagtatttga attatctaagagtatcaataaagtaccagtagatgtatctgaatc tcctggatttgtagtaaatagaatacttatacctatgataaatga agctgttggtatatatgcagatggtgttgcaagtaaagaagaaat agatgaagctatgaaattaggagcaaaccatccaatgggaccact agcattaggtgatttaatcggattagatgttgttttagctataat gaacgttttatatactgaatttggagatactaaatatagacctca tccacttttagctaaaatggttagagctaatcaattaggaagaaa aactaagataggattctatgattataataaataataagaaggaga tatacatatgagtacaagtgatgttaaagtttatgagaatgtagc tgttgaagtagatggaaatatatgtacagtgaaaatgaatagacc taaagcccttaatgcaataaattcaaagactttagaagaacttta tgaagtatttgtagatattaataatgatgaaactattgatgttgt aatattgacaggggaaggaaaggcatttgtagctggagcagatat tgcatacatgaaagatttagatgctgtagctgctaaagattttag tatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa agtagtgatagctgctgtaaacggatttgctttaggtggaggatg tgaacttgcaatggcatgtgatataagaattgcatctgctaaagc taaatttggtcagccagaagtaactcttggaataactccaggata tggaggaactcaaaggcttacaagattggttggaatggcaaaagc aaaagaattaatctttacaggtcaagttataaaagctgatgaagc tgaaaaaatagggctagtaaatagagtcgttgagccagacatttt aatagaagaagttgagaaattagctaagataatagctaaaaatgc tcagcttgcagttagatactctaaagaagcaatacaacttggtgc tcaaactgatataaatactggaatagatatagaatctaatttatt tggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagc tttcgttgaaaagagagaagctaactttataaaagggtaataaga aggagatatacatatgagaagttttgaagaagtaattaagtttgc aaaagaaagaggacctaaaactatatcagtagcatgttgccaaga taaagaagttttaatggcagttgaaatggctagaaaagaaaaaat agcaaatgccattttagtaggagatatagaaaagactaaagaaat tgcaaaaagcatagacatggatatcgaaaattatgaactgataga tataaaagatttagcagaagcatctctaaaatctgttgaattagt ttcacaaggaaaagccgacatggtaatgaaaggcttagtagacac atcaataatactaaaagcagttttaaataaagaagtaggtcttag aactggaaatgtattaagtcacgtagcagtatttgatgtagaggg atatgatagattatttttcgtaactgacgcagctatgaacttagc tcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcac agtagcacattcattagatataagtgaaccaaaagttgctgcaat atgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttga agctaaagaactagaagaaatgtatgaaagaggagaaatcaaagg ttgtatggttggtgggccttttgcaattgataatgcagtatcttt agaagcagctaaacataaaggtataaatcatcctgtagcaggacg agctgatatattattagccccagatattgaaggtggtaacatatt atataaagctttggtattcttctcaaaatcaaaaaatgcaggagt tatagttggggctaaagcaccaataatattaacttctagagcaga cagtgaagaaactaaactaaactcaatagctttaggtgttttaat ggcagcaaaggcataataagaaggagatatacatatgagcaaaat atttaaaatcttaacaataaatcctggttcgacatcaactaaaat agctgtatttgataatgaggatttagtatttgaaaaaactttaag acattcttcagaagaaataggaaaatatgagaaggtgtctgacca atttgaatttcgtaaacaagtaatagaagaagctctaaaagaagg tggagtaaaaacatctgaattagatgctgtagtaggtagaggagg acttcttaaacctataaaaggtggtacttattcagtaagtgctgc tatgattgaagatttaaaagtgggagttttaggagaacacgcttc aaacctaggtggaataatagcaaaacaaataggtgaagaagtaaa tgttccttcatacatagtagaccctgttgttgtagatgaattaga agatgttgctagaatttctggtatgcctgaaataagtagagcaag tgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgc tagagaaataaacaagaaatatgaagatataaatcttatagttgc acacatgggtggaggagtttctgttggagctcataaaaatggtaa aatagtagatgttgcaaacgcattagatggagaaggacctttctc tccagaaagaagtggtggactaccagtaggtgcattagtaaaaat gtgctttagtggaaaatatactcaagatgaaattaaaaagaaaat aaaaggtaatggcggactagttgcatacttaaacactaatgatgc tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaa attagtatatgaagctatggcatatcaaatctctaaagaaatagg agctagtgctgcagttcttaagggagatgtaaaagcaatattatt aactggtggaatcgcatattcaaaaatgtttacagaaatgattgc agatagagttaaatttatagcagatgtaaaagtttatccaggtga agatgaaatgattgcattagctcaaggtggacttagagttttaac tggtgaagaagaggctcaagtttatgataactaataa

The gene products of the bcd2-etfA3-etfB3 genes form a complex that converts crotonyl-CoA to butyryl-CoA and may exhibit dependence on oxygen as a co-oxidant. Because the recombinant bacteria of the invention are designed to produce butyrate in an oxygen-limited environment (e.g. the mammalian gut), that dependence on oxygen could have a negative effect of butyrate production in the gut. It has been shown that a single gene from Treponema denticola, trans-2-enoynl-CoA reductase (ter), can functionally replace this three gene complex in an oxygen-independent manner. Therefore, a second butyrate gene cassette in which the ter gene replaces the bcd2-etfA3-etfB3 genes of the first butyrate cassette is synthesized (Genewiz, Cambridge, Mass.). The ter gene is codon-optimized for E. coli codon usage using Integrated DNA Technologies online codon optimization tool (https://www.idtdna.com/CodonOpt). The second butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The second butyrate gene cassette is placed under control of a FNR regulatory region as described above. In certain constructs, the butyrate gene cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 20). In certain constructs, the butyrate gene cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Table 21 and Table 22).

TABLE 36 ROS regulated constructs, OxyR construct, Tet-regulated constructs Description Sequence Nucleotide ctcgagttcattatccatcctccatcgccacgatagttcatggcgataggtagaatagcaatgaacgattat sequences of ccctatcaagcattctgactgataattgctcacacgaattcattaaagaggagaaaggtaccatggatttaa pLogic031- attctaaaaaatatcagatgcttaaagagctatatgtaagcttcgctgaaaatgaagttaaacctttagcaac oxyS-butyrate agaacttgatgaagaagaaagatttccttatgaaacagtggaaaaaatggcaaaagcaggaatgatggg construct (SEQ tataccatatccaaaagaatatggtggagaaggtggagacactgtaggatatataatggcagttgaagaat ID NO: 223) tgtctagagtttgtggtactacaggagttatattatcagctcatacatctcttggctcatggcctatatatcaat atggtaatgaagaacaaaaacaaaaattcttaagaccactagcaagtggagaaaaattaggagcatttgg tcttactgagcctaatgctggtacagatgcgtctggccaacaaacaactgctgttttagacggggatgaat acatacttaatggctcaaaaatatttataacaaacgcaatagctggtgacatatatgtagtaatggcaatgac tgataaatctaaggggaacaaaggaatatcagcatttatagttgaaaaaggaactcctgggtttagctttgg agttaaagaaaagaaaatgggtataagaggttcagctacgagtgaattaatatttgaggattgcagaatac ctaaagaaaatttacttggaaaagaaggtcaaggatttaagatagcaatgtctactcttgatggtggtagaa ttggtatagctgcacaagctttaggtttagcacaaggtgctcttgatgaaactgttaaatatgtaaaagaaag agtacaatttggtagaccattatcaaaattccaaaatacacaattccaattagctgatatggaagttaaggta caagcggctagacaccttgtatatcaagcagctataaataaagacttaggaaaaccttatggagtagaag cagcaatggcaaaattatttgcagctgaaacagctatggaagttactacaaaagctgtacaacttcatgga ggatatggatacactcgtgactatccagtagaaagaatgatgagagatgctaagataactgaaatatatga aggaactagtgaagttcaaagaatggttatttcaggaaaactattaaaatagtaagaaggagatatacatat ggaggaaggatttatgaatatagtcgtttgtataaaacaagttccagatacaacagaagttaaactagatcc taatacaggtactttaattagagatggagtaccaagtataataaaccctgatgataaagcaggtttagaaga agctataaaattaaaagaagaaatgggtgctcatgtaactgttataacaatgggacctcctcaagcagatat ggctttaaaagaagctttagcaatgggtgcagatagaggtatattattaacagatagagcatttgcgggtg ctgatacttgggcaacttcatcagcattagcaggagcattaaaaaatatagattttgatattataatagctgg aagacaggcgatagatggagatactgcacaagttggacctcaaatagctgaacatttaaatcttccatcaa taacatatgctgaagaaataaaaactgaaggtgaatatgtattagtaaaaagacaatttgaagattgttgcc atgacttaaaagttaaaatgccatgccttataacaactcttaaagatatgaacacaccaagatacatgaaag ttggaagaatatatgatgctttcgaaaatgatgtagtagaaacatggactgtaaaagatatagaagttgacc cttctaatttaggtcttaaaggttctccaactagtgtatttaaatcatttacaaaatcagttaaaccagctggta caatatacaatgaagatgcgaaaacatcagctggaattatcatagataaattaaaagagaagtatatcatat aataagaaggagatatacatatgggtaacgttttagtagtaatagaacaaagagaaaatgtaattcaaact gtttctttagaattactaggaaaggctacagaaatagcaaaagattatgatacaaaagtttctgcattactttt aggtagtaaggtagaaggtttaatagatacattagcacactatggtgcagatgaggtaatagtagtagatg atgaagctttagcagtgtatacaactgaaccatatacaaaagcagcttatgaagcaataaaagcagctgac cctatagttgtattatttggtgcaacttcaataggtagagatttagcgcctagagtttctgctagaatacatac aggtcttactgctgactgtacaggtcttgcagtagctgaagatacaaaattattattaatgacaagacctgcc tttggtggaaatataatggcaacaatagtttgtaaagatttcagacctcaaatgtctacagttagaccaggg gttatgaagaaaaatgaacctgatgaaactaaagaagctgtaattaaccgtttcaaggtagaatttaatgat gctgataaattagttcaagttgtacaagtaataaaagaagctaaaaaacaagttaaaatagaagatgctaa gatattagtttctgctggacgtggaatgggtggaaaagaaaacttagacatactttatgaattagctgaaatt ataggtggagaagtttctggttctcgtgccactatagatgcaggttggttagataaagcaagacaagttggt caaactggtaaaactgtaagaccagacctttatatagcatgtggtatatctggagcaatacaacatatagct ggtatggaagatgctgagtttatagttgctataaataaaaatccagaagctccaatatttaaatatgctgatgt tggtatagttggagatgttcataaagtgcttccagaacttatcagtcagttaagtgttgcaaaagaaaaaggt gaagttttagctaactaataagaaggagatatacatatgagagaagtagtaattgccagtgcagctagaac agcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaa gaagctataaaaagagctaacataactccagatatgatagatgaatctcttttagggggagtacttacagc aggtcttggacaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctat gactataaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgc tgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggt gcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggattatcagacatatttaataactatca catgggtattactgctgaaaacatagcagagcaatggaatataactagagaagaacaagatgaattagct cttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgtt ataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatggaga aacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaataaatg atggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaact atagtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagcaactaaaaaa gctttagaagctgctaatatgactattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatc tgtagctgtaataagagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagctatagga catccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaagagaagagatgctaa aactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaagagatagtaagaagg agatatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaagt tgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaa tttaactaagttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttca actactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatataaaga aagatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaacaaatacttcatcattatcta taacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttctttaatccagttcctatg atgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatacagtatttgaattatctaag agtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgat aaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattag gagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagctataatgaa cgttttatatactgaatttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaatca attaggaagaaaaactaagataggattctatgattataataaataataagaaggagatatacatatgagtac aagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatag acctaaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatattaataatg atgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcagatattgcataca tgaaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaaa tagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatg tgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatat ggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaag ttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaa gaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaata caacttggtgctcaaactgatataaatactggaatagatatagaatctaatttatttggtctttgtttttcaacta aagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactttataaaagggtaataaga aggagatatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaactatat cagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaaaagaaaaaatagcaaat gccattttagtaggagatatagaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaatta tgaactgatagatataaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaagc cgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtct tagaactggaaatgtattaagtcacgtagcagtatttgatgtagagggatatgatagattatttttcgtaactg acgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagca cattcattagatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaa agatacagttgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttggt gggccttttgcaattgataatgcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcag gacgagctgatatattattagccccagatattgaaggtggtaacatattatataaagctttggtattcttctcaa aatcaaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttctagagcagacagtgaa gaaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataataagaaggagatatacat atgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaatagctgtatttgataatga ggatttagtatttgaaaaaactttaagacattcttcagaagaaataggaaaatatgagaaggtgtctgacca atttgaatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaattagatg ctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattcagtaagtgctgctatgattga agatttaaaagtgggagttttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggt gaagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaattagaagatgttgctagaatttc tggtatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaagatatg ctagagaaataaacaagaaatatgaagatataaatcttatagttgcacacatgggtggaggagtttctgttg gagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaaggacctttctctccagaa agaagtggtggactaccagtaggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatgaaatt aaaaagaaaataaaaggtaatggcggactagttgcatacttaaacactaatgatgctagagaagttgaag aaagaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaa taggagctagtgctgcagttcttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaa aaatgtttacagaaatgattgcagatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatga aatgattgcattagctcaaggtggacttagagttttaactggtgaagaagaggctcaagtttatgataactaa taa Nucleotide ctcgagttcattatccatcctccatcgccacgatagttcatggcgataggtagaatagcaatgaacgattat sequences of ccctatcaagcattctgactgataattgctcacacgaattcattaaagaggagaaaggtaccatgatcgtaa pLogic046- aacctatggtacgcaacaatatctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatc oxyS-butyrate agattgaatataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgttc construct (SEQ tggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggatacggggctgc ID NO: 224) gaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaataat ttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttcag acgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacag cttggccagcccagtacgtactgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaa aaacgttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgcggaaccagcaa atgacgaggaagcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaagcagc tgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccctgaagctaccc aagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggccacagcacaccgtctc aacaaagagaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgc cgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaagggcaatcatgaag gttgtattgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgat gaggaaaatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgcgttg atggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgatttcttag ctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaacgcttcgaccgtatctgataag aaggagatatacatatgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggag gagcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaa cataactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagc aagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagtttgtggtt ctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttggtgga gctgaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgct tttgttgattcaatgataaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaac atagcagagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctg aaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtga cactgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaagacctg catttaaaaaagatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagt aatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagctg gtgttgaccctaaaataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatg actattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataagagactt aaatatagatatgaataaagttaatgttaatggtggagcaatagctataggacatccaataggatgctcagg agcaagaatacttactacacttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgt ataggcggtggaatgggaactactttaatagttaagagatagtaagaaggagatatacatatgaaattagc tgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaa agagtagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagttagttactaag ggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaactactaattatgaagattta aaagatatggatttaataatagaagcatctgtagaagacatgaatataaagaaagatgttttcaagttactag atgaattatgtaaagaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttcttcta ctaagcgcccagataaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttata agtggtcagttaacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagt agatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatata tgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatggg accactagcattaggtgatttaatcggattagatgttgttttagctataatgaacgttttatatactgaatttgga gatactaaatatagacctcatccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaag ataggattctatgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagtttatga gaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaat aaattcaaagactttagaagaactttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaata ttgacaggggaaggaaaggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagc tgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagc tgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgct aaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggaggaactcaaaggcttac aagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctg aaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagctaag ataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgat ataaatactggaatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaat gtcagctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgagaa gttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaagata aagaagttttaatggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtaggagatata gaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaattatgaactgatagatataaaag atttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaaggct tagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtcttagaactggaaatgtattaa gtcacgtagcagtatttgatgtagagggatatgatagattatttttcgtaactgacgcagctatgaacttagct cctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagcacattcattagatataagtgaa ccaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaa gaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataa tgcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcaggacgagctgatatattatta gccccagatattgaaggtggtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggag ttatagttggggctaaagcaccaataatattaacttctagagcagacagtgaagaaactaaactaaactca atagctttaggtgttttaatggcagcaaaggcataataagaaggagatatacatatgagcaaaatatttaaa atcttaacaataaatcctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaa actttaagacattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaa gtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagaggag gacttcttaaacctataaaaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagtt ttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaatgttccttc atacatagtagaccctgttgttgtagatgaattagaagatgttgctagaatttctggtatgcctgaaataagta gagcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaacaagaa atatgaagatataaatcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaatggtaaa atagtagatgttgcaaacgcattagatggagaaggacctttctctccagaaagaagtggtggactaccagt aggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaa tggcggactagttgcatacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtgatg aaaaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctgcagttc ttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaaaaatgtttacagaaatgattgc agatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctcaaggt ggacttagagttttaactggtgaagaagaggctcaagtttatgataactaataa Nucleotide ctcgagatgctagcaattgtgagcggataacaattgacattgtgagcggataacaagatactgagcacat sequences of cagcaggacgcactgaccttaattaaaagaattcattaaagaggagaaaggtaccatgaatattcgtgatc pZA22-oxyR ttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgccacgttagccag construct (SEQ ccgacgcttagcgggcaaattcgtaagctggaagatgagctgggcgtgatgttgctggagcggaccag ID NO: 225) ccgtaaagtgttgttcacccaggcgggaatgctgctggtggatcaggcgcgtaccgtgctgcgtgaggt gaaagtccttaaagagatggcaagccagcagggcgagacgatgtccggaccgctgcacattggtttgat tcccacagttggaccgtacctgctaccgcatattatccctatgctgcaccagacctttccaaagctggaaat gtatctgcatgaagcacagacccaccagttactggcgcaactggacagcggcaaactcgattgcgtgat cctcgcgctggtgaaagagagcgaagcattcattgaagtgccgttgtttgatgagccaatgttgctggcta tctatgaagatcacccgtgggcgaaccgcgaatgcgtaccgatggccgatctggcaggggaaaaactg ctgatgctggaagatggtcactgtttgcgcgatcaggcaatgggtttctgttttgaagccggggcggatga agatacacacttccgcgcgaccagcctggaaactctgcgcaacatggtggcggcaggtagcgggatca ctttactgccagcgctggctgtgccgccggagcgcaaacgcgatggggttgtttatctgccgtgcattaa gccggaaccacgccgcactattggcctggtttatcgtcctggctcaccgctgcgcagccgctatgagca gctggcagaggccatccgcgcaagaatggatggccatttcgataaagttttaaaacaggcggtttaagga tcccatggtacgcgtgctagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt atctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctaggggatatattcc gcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggcttacgaacgg ggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagcc gtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaac ccgacaggactataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgccttt cggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtag gcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccggta actatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatt tagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgct cctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaag gcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaatc agataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagccccatacgatat aagttgttactagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatc cagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctctcgaaccccagagtc ccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgatacc gtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacg ctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccatttt ccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgc gccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcga caagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggt agccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaag gtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgac aacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcc tgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacag ccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccac ccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacgatcctcatcctgtctctt gatcagatcttgatcccctgcgccatcagatccttggcggcaagaaagccatccagtttactttgcagggc ttcccaaccttaccagagggcgccccagctggcaattccgacgtctaagaaaccattattatcatgacatt aacctataaaaataggcgtatcacgaggccctttcgtcttcac Nucleotide gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgtttttctaatccgcatatgatca sequences of attcaaggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatg pLogic031-tet- gcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac butyrate ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaa construct (SEQ aggctaattgattttcgagagtttcatactgtttttctgtaggccgt gtacctaaatgtacttttgctccatcgcg ID NO: 226) The atgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaag sequence ggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattatta encoding TetR is catgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattcc underlined, and gacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt the overlapping

tetR/tetA attttgtttaactttaagaaggagatatacatatggatttaaattctaaaaaatatcagatgcttaaagagctat promoters are atgtaagcttcgctgaaaatgaagttaaacctttagcaacagaacttgatgaagaagaaagatttccttatg

aaacagtggaaaaaatggcaaaagcaggaatgatgggtataccatatccaaaagaatatggtggagaa ggtggagacactgtaggatatataatggcagttgaagaattgtctagagtttgtggtactacaggagttata ttatcagctcatacatctcttggctcatggcctatatatcaatatggtaatgaagaacaaaaacaaaaattctt aagaccactagcaagtggagaaaaattaggagcatttggtcttactgagcctaatgctggtacagatgcgt ctggccaacaaacaactgctgttttagacggggatgaatacatacttaatggctcaaaaatatttataacaa acgcaatagctggtgacatatatgtagtaatggcaatgactgataaatctaaggggaacaaaggaatatc agcatttatagttgaaaaaggaactcctgggtttagctttggagttaaagaaaagaaaatgggtataagag gttcagctacgagtgaattaatatttgaggattgcagaatacctaaagaaaatttacttggaaaagaaggtc aaggatttaagatagcaatgtctactcttgatggtggtagaattggtatagctgcacaagctttaggtttagc acaaggtgctcttgatgaaactgttaaatatgtaaaagaaagagtacaatttggtagaccattatcaaaattc caaaatacacaattccaattagctgatatggaagttaaggtacaagcggctagacaccttgtatatcaagc agctataaataaagacttaggaaaaccttatggagtagaagcagcaatggcaaaattatttgcagctgaaa cagctatggaagttactacaaaagctgtacaacttcatggaggatatggatacactcgtgactatccagta gaaagaatgatgagagatgctaagataactgaaatatatgaaggaactagtgaagttcaaagaatggttat ttcaggaaaactattaaaatagtaagaaggagatatacatatggaggaaggatttatgaatatagtcgtttgt ataaaacaagttccagatacaacagaagttaaactagatcctaatacaggtactttaattagagatggagta ccaagtataataaaccctgatgataaagcaggtttagaagaagctataaaattaaaagaagaaatgggtg ctcatgtaactgttataacaatgggacctcctcaagcagatatggctttaaaagaagctttagcaatgggtg cagatagaggtatattattaacagatagagcatttgcgggtgctgatacttgggcaacttcatcagcattag caggagcattaaaaaatatagattttgatattataatagctggaagacaggcgatagatggagatactgca caagttggacctcaaatagctgaacatttaaatcttccatcaataacatatgctgaagaaataaaaactgaa ggtgaatatgtattagtaaaaagacaatttgaagattgttgccatgacttaaaagttaaaatgccatgccttat aacaactcttaaagatatgaacacaccaagatacatgaaagttggaagaatatatgatgctttcgaaaatg atgtagtagaaacatggactgtaaaagatatagaagttgacccttctaatttaggtcttaaaggttctccaac tagtgtatttaaatcatttacaaaatcagttaaaccagctggtacaatatacaatgaagatgcgaaaacatca gctggaattatcatagataaattaaaagagaagtatatcatataataagaaggagatatacatatgggtaac gttttagtagtaatagaacaaagagaaaatgtaattcaaactgtttctttagaattactaggaaaggctacag aaatagcaaaagattatgatacaaaagtttctgcattacttttaggtagtaaggtagaaggtttaatagatac attagcacactatggtgcagatgaggtaatagtagtagatgatgaagctttagcagtgtatacaactgaac catatacaaaagcagcttatgaagcaataaaagcagctgaccctatagttgtattatttggtgcaacttcaat aggtagagatttagcgcctagagtttctgctagaatacatacaggtcttactgctgactgtacaggtcttgca gtagctgaagatacaaaattattattaatgacaagacctgcctttggtggaaatataatggcaacaatagttt gtaaagatttcagacctcaaatgtctacagttagaccaggggttatgaagaaaaatgaacctgatgaaact aaagaagctgtaattaaccgtttcaaggtagaatttaatgatgctgataaattagttcaagttgtacaagtaat aaaagaagctaaaaaacaagttaaaatagaagatgctaagatattagtttctgctggacgtggaatgggtg gaaaagaaaacttagacatactttatgaattagctgaaattataggtggagaagtttctggttctcgtgccac tatagatgcaggttggttagataaagcaagacaagttggtcaaactggtaaaactgtaagaccagaccttt atatagcatgtggtatatctggagcaatacaacatatagctggtatggaagatgctgagtttatagttgctat aaataaaaatccagaagctccaatatttaaatatgctgatgttggtatagttggagatgttcataaagtgcttc cagaacttatcagtcagttaagtgttgcaaaagaaaaaggtgaagttttagctaactaataagaaggagat atacatatgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggaggagcattta aatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactcc agatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaagacaaat agcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagtttgtggttctggattaag atctgtttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaaca tgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattca atgataaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaacatagcagag caatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctc aagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagta gataaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaa gatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagtaatggctaaa gaaaaagctgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccct aaaataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatgactattgaagat atagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataagagacttaaatatagatat gaataaagttaatgttaatggtggagcaatagctataggacatccaataggatgctcaggagcaagaata cttactacacttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgtataggcggtg gaatgggaactactttaatagttaagagatagtaagaaggagatatacatatgaaattagctgtaataggta gtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagagtagaac tcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagttagttactaagggaaaaatgg atgaagctacaaaagcagaaatattaagtcatgttagttcaactactaattatgaagatttaaaagatatgga tttaataatagaagcatctgtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgta aagaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttcttctactaagcgccc agataaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggtcagtt aacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgtatctga atctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatatatgcagatggtgt tgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccactagcatta ggtgatttaatcggattagatgttgttttagctataatgaacgttttatatactgaatttggagatactaaatata gacctcatccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattctatg attataataaataataagaaggagatatacatatgagtacaagtgatgttaaagtttatgagaatgtagctgtt gaagtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagac tttagaagaactttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaatattgacagggga aggaaaggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattt tagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacg gatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgctaaagctaaattt ggtcagccagaagtaactcttggaataactccaggatatggaggaactcaaaggcttacaagattggttg gaatggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaaatagg gctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagctaagataatagctaa aaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgatataaatactgg aatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgtt gaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgagaagttttgaagaag taattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaagataaagaagttttaa tggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtaggagatatagaaaagactaa agaaattgcaaaaagcatagacatggatatcgaaaattatgaactgatagatataaaagatttagcagaag catctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaaggcttagtagacacat caataatactaaaagcagttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtagcag tatttgatgtagagggatatgatagattatttttcgtaactgacgcagctatgaacttagctcctgatacaaat actaaaaagcaaatcatagaaaatgcttgcacagtagcacattcattagatataagtgaaccaaaagttgct gcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactagaagaa atgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataatgcagtatctttag aagcagctaaacataaaggtataaatcatcctgtagcaggacgagctgatatattattagccccagatattg aaggtggtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggagttatagttggggct aaagcaccaataatattaacttctagagcagacagtgaagaaactaaactaaactcaatagctttaggtgtt ttaatggcagcaaaggcataataagaaggagatatacatatgagcaaaatatttaaaatcttaacaataaat cctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaactttaagacattct tcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaagaagc tctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagaggaggacttcttaaaccta taaaaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagttttaggagaacacg cttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaatgttccttcatacatagtagac cctgttgttgtagatgaattagaagatgttgctagaatttctggtatgcctgaaataagtagagcaagtgtag tacatgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatata aatcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaatggtaaaatagtagatgttg caaacgcattagatggagaaggacctttctctccagaaagaagtggtggactaccagtaggtgcattagt aaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcggactag ttgcatacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaa ttagtatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctgcagttcttaagggagat gtaaaagcaatattattaactggtggaatcgcatattcaaaaatgtttacagaaatgattgcagatagagtta aatttatagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctcaaggtggacttagagt tttaactggtgaagaagaggctcaagtttatgataactaataa Nucleotide gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgtttttctaatccgcatatgatca sequences of attcaaggccgaataagaaggctggctctgcaccaggtgatcaaataattcgatagatgtcgtaataatg pLogic046-tet- gcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac butyrate ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaa construct (SEQ aggctaattgattacgagagatcatactgatactgtaggccgtgtacctaaatgtacattgctccatcgce ID NO: 227) The atgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaag sequence ggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattatta encoding TetR is catgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattcc underlined, and gacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt the overlapping

tetR/tetA attttgtttaactttaagaaggagatatacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaac promoters are gcccatcctcagggctgcaagaagggagtggaagatcagattgaatataccaagaaacgcattaccgc

agaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaatggttacggcctg gcgagccgcattactgctgcgttcggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggtt cagaaaccaaatatggtacaccgggatggtacaataatttggcatttgatgaagcggcaaaacgcgagg gtctttatagcgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattgaggaagc caaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgatac aggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatccgttta ctggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaagcagccgccactgttaaagtt atggggggtgaagattgggaacgttggattaagcagctgtcgaaggaaggcctcttagaagaaggctgt attaccttggcctatagttatattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaag gccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcgt gagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccag cttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccg agcgcctgtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgattgggag ttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatc tctcactgacttagcggggtaccgccatgatttcttagctagtaacggctttgatgtagaaggtattaattatg aagcggaagttgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgc cagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagttaggg gtaacagcagctaaagaagctataaaaagagctaacataactccagatatgatagatgaatctcttttagg gggagtacttacagcaggtcttggacaaaatatagcaagacaaatagcattaggagcaggaataccagt agaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttat agcattaggtgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaa gtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggattatcagacat atttaataactatcacatgggtattactgctgaaaacatagcagagcaatggaatataactagagaagaac aagatgaattagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaaga aatagttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctgg cactacaatggagaaacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatg catcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactaggaata gagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttc cagcaactaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttgaagctaatgagg catttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgttaatggtgga gcaatagctataggacatccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaag agaagagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaag agatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgt acaaacttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatgttta gctttattagataaaaatttaactaagttagttactaagggaaaaatggatgaagctacaaaagcagaaata ttaagtcatgttagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaa gacatgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaacaa atacttcatcattatctataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttct ttaatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatacag tatttgaattatctaagagtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaatagaa tacttatacctatgataaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatga agctatgaaattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgt tttagctataatgaacgttttatatactgaatttggagatactaaatatagacctcatccacttttagctaaaatg gttagagctaatcaattaggaagaaaaactaagataggattctatgattataataaataataagaaggagat atacatatgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtacag tgaaaatgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgt agatattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagc agatattgcatacatgaaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttg gagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaa cttgcaatggcatgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactcttgga ataactccaggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaa tctttacaggtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagcca gacattttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttagatact ctaaagaagcaatacaacttggtgctcaaactgatataaatactggaatagatatagaatctaatttatttggt ctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactttataa aagggtaataagaaggagatatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagagg acctaaaactatatcagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaaaag aaaaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaattgcaaaaagcatagacat ggatatcgaaaattatgaactgatagatataaaagatttagcagaagcatctctaaaatctgttgaattagttt cacaaggaaaagccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcagttttaaat aaagaagtaggtcttagaactggaaatgtattaagtcacgtagcagtatttgatgtagagggatatgataga ttatttttcgtaactgacgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatg cttgcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagta aatccaaaaatgaaagatacagttgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaa ggttgtatggttggtgggccttttgcaattgataatgcagtatctttagaagcagctaaacataaaggtataa atcatcctgtagcaggacgagctgatatattattagccccagatattgaaggtggtaacatattatataaagc tttggtattcttctcaaaatcaaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttcta gagcagacagtgaagaaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataataa gaaggagatatacatatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaata gctgtatttgataatgaggatttagtatttgaaaaaactttaagacattcttcagaagaaataggaaaatatga gaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaa acatctgaattagatgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattcagtaa gtgctgctatgattgaagatttaaaagtgggagttttaggagaacacgcttcaaacctaggtggaataatag caaaacaaataggtgaagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaattagaag atgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaaaaggcaat agcaagaagatatgctagagaaataaacaagaaatatgaagatataaatcttatagttgcacacatgggtg gaggagtttctgttggagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaagga cctttctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgctttagtggaaaatat actcaagatgaaattaaaaagaaaataaaaggtaatggcggactagttgcatacttaaacactaatgatgc tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggcatatc aaatctctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaagcaatattattaactggtg gaatcgcatattcaaaaatgtttacagaaatgattgcagatagagttaaatttatagcagatgtaaaagtttat ccaggtgaagatgaaatgattgcattagctcaaggtggacttagagttttaactggtgaagaagaggctca agtttatgataactaataa

In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter.

In a third butyrate gene cassette, the pbt and buk genes are replaced with tesB. TesB is a thioesterase found in E. Coli that cleaves off the butyrate from butyryl-coA, thus obviating the need for pbt-buk.

In one embodiment, the tesB cassette is placed under the control of a FNR regulatory region selected from any of the sequences in Table 6. In an alternate embodiment, the tesB cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR. In yet another embodiment, the tesB cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR. In certain constructs, the different described butyrate gene cassettes are each placed under the control of a tetracycline-inducible or constitutive promoter. For example, genetically engineered Nissle are generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB expressed under the control of a nitric oxide-responsive regulatory element. SEQ ID NO: 228 comprises a reverse complement of the nsrR repressor gene from Neisseria gonorrhoeae (underlined), intergenic region containing divergent promoters controlling nsrR and the butyrogenic gene cassette and their respective RBS (bold), and the butyrate genes (ter-thiA-hbd-crt-tesB) separated by RBS.

TABLE 37 SEQ ID NO: 228 ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggctt gttgagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgc accgccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgg gcccatacactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatatt gatgcgttcgggcggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtg caagaacccgcctttgaccagcgcggtaaccactttcatcaaatggcttttggaaat gccgtaggtcgaggcgatggtggcgatattgaccagcgcgtcgtcgttgacggcggt gtagatgaggacgcgcagcccgtagtcggtatgttgggtcagatacat acaacctcc ttagtacatgcaaaattatttctagagcaacatacgagccggaagcataaagtgtaa agcctggggtgcctaatgagttgagttgaggaattataacaggaagaaatattcctc atacgcttgtaattcctctatggttgttgacaattaatcatcggctcgtataatgta taacattcatattttgtgaattttaaactctagaaataattttgtttaactttaaga aggagatatacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgc ccatcctcagggctgcaagaagggagtggaagatcagattgaatataccaagaaacg cattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttgg ctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggatacggggc tgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacacc gggatggtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttatag cgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattgagga agccaaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagt acgtactgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaa aacgttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgc ggaaccagcaaatgacgaggaagcagccgccactgttaaagttatggggggtgaaga ttgggaacgttggattaagcagctgtcgaaggaaggcctcttagaagaaggctgtat taccttggcctatagttatattggccctgaagctacccaagctttgtaccgtaaagg cacaatcggcaaggccaaagaacacctggaggccacagcacaccgtctcaacaaaga gaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaag cgccgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaaga gaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcct gtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatga ttgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagtcac gggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgatttcttagc tagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaacgcttcga ccgtatctgataagaaggagatatacatatgagagaagtagtaattgccagtgcagc tagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagtt aggggtaacagcagctaaagaagctataaaaagagctaacataactccagatatgat agatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaag acaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatat agtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattagg tgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttattt agtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaat gataaaagatggattatcagacatatttaataactatcacatgggtattactgctga aaacatagcagagcaatggaatataactagagaagaacaagatgaattagctcttgc aagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagt tcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatat taagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaaga tggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagt agtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatagt ttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagc aactaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttga agctaatgaggcatttgctgcccaatctgtagctgtaataagagacttaaatataga tatgaataaagttaatgttaatggtggagcaatagctataggacatccaataggatg ctcaggagcaagaatacttactacacttttatatgaaatgaagagaagagatgctaa aactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaa gagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactat gggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagag tagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaa gttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgt tagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatc tgtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgtaa agaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttc ttctactaagcgcccagataaagttataggaatgcatttctttaatccagttcctat gatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatac agtatttgaattatctaagagtatcaataaagtaccagtagatgtatctgaatctcc tggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatata tgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaa ccatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagc tataatgaacgttttatatactgaatttggagatactaaatatagacctcatccact tttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattcta tgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagt ttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatag acctaaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatt tgtagatattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaa ggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgc taaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa agtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaat ggcatgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaac tcttggaataactccaggatatggaggaactcaaaggcttacaagattggttggaat ggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctga aaaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttga gaaattagctaagataatagctaaaaatgctcagcttgcagttagatactctaaaga agcaatacaacttggtgctcaaactgatataaatactggaatagatatagaatctaa tttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgt tgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgAG TCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGAAAAAATTGAGGAAGGACT CTTTCGCGGCCAGAGTGAAGATTTAGGTTTACGCCAGGTGTTTGGCGGCCAGGTCGT GGGTCAGGCCTTGTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGATTATTTATGATGT CGAAACGCTGCGTGACGGTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAAAA CGGCAAACCGATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGA ACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCGGAAACGCA AATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGATAAATTCATCTG CGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCCACTGAAAGGTCACGT CGCAGAACCACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGACCT GCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTAACTTCCTGCCGGTAGC TCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTGCCACCATTGA CCATTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGT GGAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATACCCA AGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTAATCACAATta a

Example 11 Construction of Vectors for Overproducing Butyrate Using a tet-Inducible Promoter

To facilitate inducible production of butyrate in Escherichia coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile (bcd, etfB, etfA, thiA, hbd, crt, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen, Cambridge, Mass.) and cloned into vector pBR to create pLogic. As synthesized, the genes were placed under control of a tetracycline-inducible promoter, with the tet repressor (tetR) expressed constitutively, divergent from the tet-inducible synthetic butyrate operon. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a base pair ribosome binding site derived from the T promoter.

The gene products of bcd-etfA-etfB form a complex that convert crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co-oxidant. Because an effective probiotic should be able to function in an oxygen-limited environment (e.g. the mammalian gut), and because it has been shown that a single gene from Treponema denticola can functionally replace this three gene complex in an oxygen-independent manner (trans--enoynl-CoA reductase; ter), we created a second plasmid capable of butyrate production in E. coli . Inverse PCR was used to amplify the entire sequence of pLogic outside of the bcd-etfA-etfB region. The ter gene was codon optimized for E. coli codon usage using Integrated DNA technologies online codon optimization tool (https://www.idtdna.com/CodonOpt), synthesized (Genewiz, Cambridge, Mass.), and cloned into this inverse PCR fragment using Gibson assembly to create pLogic.

Example 12 Transforming E. coli

Each plasmid is transformed into E. coli Nissle or E. coli DH5a. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli Nissle or E. coli DH5a is diluted 1:100 in 5 mL of lysogeny broth (LB) and grown until it reached an OD₆₀₀ of 0.4-0.6. The cell culture medium contains a selection marker, e.g., ampicillin, that is suitable for the plasmid. The E. coli cells are then centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 1 mL of 4° C. water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C. water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are finally resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 0.5 μg of one of the above plasmids is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. One mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C. for 1 hr. The cells are spread out on an LB plate containing ampicillin and incubated overnight.

In alternate embodiments, the butyrate cassette can be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, Mass.). Organization of the constructs and nucleotide sequences are provided herein. To create a vector capable of integrating the synthesized butyrate cassette construct into the chromosome, Gibson assembly was first used to add 1000 bp sequences of DNA homologous to the Nissle lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the lacZ locus in the Nissle genome. Gibson assembly was used to clone the fragment between these arms. PCR was used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the butyrate cassette between them. This PCR fragment was used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells were grown out for 2 hours before plating on chloramphenicol at 20 ug/mL at 37 degrees C. Growth at 37 degrees C. also cures the pKD46 plasmid. Transformants containing cassette were chloramphenicol resistant and lac-minus (lac−).

Example 13 Production of Butyrate in Recombinant E. coli

Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hrs) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

Example 14 Production of Butyrate in Recombinant E. coli

Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hrs) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

Example 15 Production of Butyrate in Recombinant E. coli Using Tet-Inducible Promoter

FIG. 2 shows butyrate cassettes described above under the control of a tet-inducible promoter. Production of butyrate is assessed using the methods described below in Example 21. The tet-inducible cassettes tested include (1) tet-butyrate cassette comprising all eight genes (pLOGIC031); (2) tet-butyrate cassette in which the ter is substituted (pLOGIC046) and (3) tet-butyrate cassette in which tesB is substituted in place of pbt and buk genes.

FIG. 6A shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen, in which there is no significant difference in butyrate production. Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous E. Coli tesB gene (a thioesterase that cleaves off the butyrate portion from butyryl CoA).

Overnight cultures of cells were diluted 1:100 in Lb and grown for 1.5 hours until early log phase was reached at which point anhydrous tet was added at a final concentration of 100 ng/m1 to induce plasmid expression. After 2 hours induction, cells were washed and resuspended in M9 minimal media containing 0.5% glucose at OD600=0.5. Samples were removed at indicated times and cells spun down. The supernatant was tested for butyrate production using LC-MS. FIG. 6B shows butyrate production in strains comprising a tet-butyrate cassette having ter substitution (pLOGIC046) or the tesB substitution (ptb-buk deletion), demonstrating that the tesB substituted strain has greater butyrate production.

FIG. 7 shows the BW25113 strain of E. Coli, which is a common cloning strain and the background of the KEIO collection of E. Coli mutants. NuoB mutants having NuoB deletion were obtained. NuoB is a protein complex involved in the oxidation of NADH during respiratory growth (form of growth requiring electron transport). Preventing the coupling of NADH oxidation to electron transport allows an increase in the amount of NADH being used to support butyrate production. FIG. 7 shows that compared with wild-type Nissle, deletion of NuoB results in grater production of butyrate.

TABLE 38 pLOGIC046-tesB-butyrate (SEQ ID NO: 229) pLOGIC046-tesB-butyrate: SEQ ID NO: 229 gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgttt ttctaatccgcatatgatcaattcaaggccgaataagaaggctggctctgcaccttggt gatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtt tccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaa atgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata aaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaa tgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttatt acgtaaaaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctat ctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgat tccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctag acatcattaattcctaatttttgttgacactctatcattgatagagttattttaccact ccctatcagtgatagagaaaagtgaactctagaaataattttgtttaactttaagaagg agatatacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatc ctcagggctgcaagaagggagtggaagatcagattgaatataccaagaaacgcattacc gcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaa tggttacggcctggcgagccgcattactgctgcgttcggatacggggctgcgaccatcg gcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaat aatttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacgg cgatgcgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggta tcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgataca ggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagt agatccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaag cagccgccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctg tcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccc tgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctgg aggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtg aataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgc cagcttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatca cgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaa aatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgc gttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtacc gccatgatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaa gttgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaat tgccagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcag cggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactcca gatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatat agcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataa atatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcatta ggtgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttattt agtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatga taaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaac atagcagagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtca aaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttg ttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctggc actacaatggagaaacttgctaagttaagacctgcatttaaaaaagatggaacagttac tgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagtaatggctaaag aaaaagctgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagct ggtgttgaccctaaaataatgggatatggaccagttccagcaactaaaaaagctttaga agctgctaatatgactattgaagatatagatttagttgaagctaatgaggcatttgctg cccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgttaat ggtggagcaatagctataggacatccaataggatgctcaggagcaagaatacttactac acttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgtatag gcggtggaatgggaactactttaatagttaagagatagtaagaaggagatatacatatg aaattagctgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaag ttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatgtttag ctttattagataaaaatttaactaagttagttactaagggaaaaatggatgaagctaca aaagcagaaatattaagtcatgttagttcaactactaattatgaagatttaaaagatat ggatttaataatagaagcatctgtagaagacatgaatataaagaaagatgttttcaagt tactagatgaattatgtaaagaagatactatcttggcaacaaatacttcatcattatct ataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttctt taatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaag ttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgta tctgaatctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgt tggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattag gagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgtt ttagctataatgaacgttttatatactgaatttggagatactaaatatagacctcatcc acttttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattct atgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagtt tatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacc taaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtag atattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaaggcattt gtagctggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattt tagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatag ctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatata agaattgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataactcc aggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaat taatctttacaggtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaat agagtcgttgagccagacattttaatagaagaagttgagaaattagctaagataatagc taaaaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaa ctgatataaatactggaatagatatagaatctaatttatttggtctttgtttttcaact aaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactttataaa agggtaataagaaggagatatacatatgAGTCAGGCGCTAAAAAATTTACTGACATTGT TAAATCTGGAAAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTTTA CGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCAAAAGAGACCGT CCCTGAAGAGCGGCTGGTACATTCGTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTA AGAAGCCGATTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGCGCCCGC CGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTATATGACTGCCTCTTTCCAGGC ACCAGAAGCGGGTTTCGAACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCC TCCCTTCGGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAA GATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCCACT GAAAGGTCACGTCGCAGAACCACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGC CGGATGACCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTAACTTCCTG CCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTGCCAC CATTGACCATTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATA GCGTGGAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATACC CAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTAATCACAATta a

Example 16 Production of Butyrate in Recombinant E. coli

Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N₂, 5% CO₂, 5%H₂). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

In an alternate embodiment, overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) was added to cultures at a final concentration of 0.3 mM to induce expression from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. Genetically engineered Nissle comprising pLogic031-nsrR-norB-butyrate operon construct; SYN507) or (pLogic046-nsrR-norB-butyrate operon construct; SYN--508) produce significantly more butyrate as compared to wild-type Nissle.

Genetically engineered Nissle were generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB (SEQ ID NO: 15) expressed under the control of a tetracycline promoter (pLOGIC046-tesB-butyrate; SEQ ID NO: 208). SEQ ID NO: 208 comprises a reverse complement of the tetR repressor (underlined), an intergenic region containing divergent promoters controlling tetR and the butyrate operon and their respective RBS (bold), and the butyrate genes (ter-thiAl-hbd-crt2-tesB) separated by RBS.

Overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, anhydrous tetracycline (ATC) was added to cultures at a final concentration of 100 ng/mL to induce expression of butyrate genes from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. Replacement of pbt and buk with tesB leads to greater levels of butyrate production.

FIG. 8C shows butyrate production in strains comprising an FNR-butyrate cassette SYN501 (having the ter substitution) in the presence/absence of glucose and oxygen. FIG. 8C shows that bacteria need both glucose and anaerobic conditions for butyrate production from the FNR promoter. Cells were grown aerobically or anaerobically in media containing no glucose (LB) or in media containing glucose at 0.5% (RMC). Culture samples were taken at indicated time pints and supernatant fractions were assessed for butyrate concentration using LC-MS. These data show that SYN501 requires glucose for butyrate production and that in the presence of glucose butyrate production can be enhanced under anaerobic conditions when under the control of the anaerobic FNR-regulated ydfZ promoter.

TABLE 39 Butyrate cassette sequences SEQ ID Description Sequence NO ydfZ + RBS CATTTCCTCTCATCCCATCCGGGGTGAGAGT SEQ ID (RBS is bolded) CTTTTCCCCCGACTTATGGCTCATGCATGCAT NO: 230 CAAAAAAGATGTGAGCTTGATCAAAAACAA AAAATATTTCACTCGACAGGAGTATTTATAT TGCGCCCGGATCCCTCTAGAAATAATTTTGT TTAACTTTAAGAAGGAGATATACAT First RBS (in TTTGTTTAACTTTAAGAAGGAGA SEQ ID ydfZ = RBS) NO: 231 Internal RBS taagaaggagatatacat SEQ ID between genes NO: 211 Butyrate cassette CATTTCCTCTCATCCCATCCGGGGTGAGAGT SEQ ID under the control CTTTTCCCCCGACTTATGGCTCATGCATGCAT NO: 232 of the ydfZ CAAAAAAGATGTGAGCTTGATCAAAAACAA promoter AAAATATTTCACTCGACAGGAGTATTTATAT (uppercase: ydfZ TGCGCCCGGATCCCTCTAGAAATAATTTTGT promoter, with TTAACTTTAAGAAGGAGATATACATatgatcgt RBS in bold; aaaacctatggtacgcaacaatatctgcctgaacgcccatcctcagggct lower case: gcaagaagggagtggaagatcagattgaatataccaagaaacgcattac coding regions cgcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgctt in the following ggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcgg order: ter, thiA, atacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaa hbd, crt2, pbt, accaaatatggtacaccgggatggtacaataatttggcatttgatgaagcg buk, separated gcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttc by internal RBS agacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtat (uppercase and caaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcct underlined) gatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttc acaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccg cggaaccagcaaatgacgaggaagcagccgccactgttaaagttatgg ggggtgaagattgggaacgttggattaagcagctgtcgaaggaaggcct cttagaagaaggctgtattaccttggcctatagttatattggccctgaagct acccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacc tggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtg ccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaat cccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaa gggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagc gcctgtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattc gcattgatgattgggagttagaagaagacgtccagaaagcggtatccgc gttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttag cggggtaccgccatgatttcttagctagtaacggctttgatgtagaaggtat taattatgaagcggaagttgaacgcttcgaccgtatctgaTAAGAA GGAGATATACATatgagagaagtagtaattgccagtgcagct agaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggta gagttaggggtaacagcagctaaagaagctataaaaagagctaacataa ctccagatatgatagatgaatctcttttagggggagtacttacagcaggtct tggacaaaatatagcaagacaaatagcattaggagcaggaataccagta gaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgt ttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttg gtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcgagata tggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatgga ttatcagacatatttaataactatcacatgggtattactgctgaaaacatagc agagcaatggaatataactagagaagaacaagatgaattagctcttgcaa gtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaa atagttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaa gatgaatatattaagcctggcactacaatggagaaacttgctaagttaaga cctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaata aatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaa ctaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttg accctaaaataatgggatatggaccagttccagcaactaaaaaagctttag aagctgctaatatgactattgaagatatagatttagttgaagctaatgaggc atttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaata aagttaatgttaatggtggagcaatagctataggacatccaataggatgct caggagcaagaatacttactacacttttatatgaaatgaagagaagagatg ctaaaactggtcttgctacactttgtataggcggtggaatgggaactacttt aatagttaagagatagTAAGAAGGAGATATACATatgaa attagctgtaataggtagtggaactatgggaagtggtattgtacaaactttt gcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctata gataaatgtttagctttattagataaaaatttaactaagttagttactaaggga aaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaact actaattatgaagatttaaaagatatggatttaataatagaagcatctgtaga agacatgaatataaagaaagatgttttcaagttactagatgaattatgtaaa gaagatactatcttggcaacaaatacttcatcattatctataacagaaatag cttcttctactaagcgcccagataaagttataggaatgcatttctttaatcca gttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagt tacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtaga tgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaa tgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaataga tgaagctatgaaattaggagcaaaccatccaatgggaccactagcattag gtgatttaatcggattagatgttgttttagctataatgaacgttttatatactga atttggagatactaaatatagacctcatccacttttagctaaaatggttagag ctaatcaattaggaagaaaaactaagataggattctatgattataataaata aTAAGAAGGAGATATACATatgagtacaagtgatgttaa agtttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaa atgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaa ctttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaatatt gacaggggaaggaaaggcatttgtagctggagcagatattgcatacatg aaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaaag cttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacg gatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaat tgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataact ccaggatatggaggaactcaaaggcttacaagattggttggaatggcaa aagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctg aaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaag aagttgagaaattagctaagataatagctaaaaatgctcagcttgcagtta gatactctaaagaagcaatacaacttggtgctcaaactgatataaatactg gaatagatatagaatctaatttatttggtctttgatttcaactaaagaccaaaa agaaggaatgtcagctttcgttgaaaagagagaagctaactttataaaag ggtaaTAAGAAGGAGATATACATatgagaagttttgaag aagtaattaagtttgcaaaagaaagaggacctaaaactatatcagtagcat gttgccaagataaagaagttttaatggcagttgaaatggctagaaaagaa aaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaatt gcaaaaagcatagacatggatatcgaaaattatgaactgatagatataaaa gatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaag ccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagca gttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtag cagtatttgatgtagagggatatgatagattatttttcgtaactgacgcagct atgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgctt gcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatat gcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaa agaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttg gtgggccttttgcaattgataatgcagtatctttagaagcagctaaacataa aggtataaatcatcctgtagcaggacgagctgatatattattagccccaga tattgaaggtggtaacatattatataaagctttggtattcttctcaaaatcaaa aaatgcaggagttatagttggggctaaagcaccaataatattaacttctag agcagacagtgaagaaactaaactaaactcaatagctttaggtgttttaatg gcagcaaaggcataaTAAGAAGGAGATATACATatga gcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaata gctgtatttgataatgaggatttagtatttgaaaaaactttaagacattcttca gaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaa caagtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaatt agatgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtac ttattcagtaagtgctgctatgattgaagatttaaaagtgggagttttaggag aacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaaga agtaaatgttccttcatacatagtagaccctgttgttgtagatgaattagaag atgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtaca tgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaaca agaaatatgaagatataaatcttatagttgcacacatgggtggaggagtttc tgttggagctcataaaaatggtaaaatagtagatgttgcaaacgcattagat ggagaaggacctttctctccagaaagaagtggtggactaccagtaggtg cattagtaaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaa gaaaataaaaggtaatggcggactagttgcatacttaaacactaatgatgc tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaattag tatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctg cagttcttaagggagatgtaaaagcaatattattaactggtggaatcgcata ttcaaaaatgtttacagaaatgattgcagatagagttaaatttatagcagatg taaaagtttatccaggtgaagatgaaatgattgcattagctcaaggtggact tagagttttaactggtgaagaagaggctcaagtttatgataactaataa

In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 232 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 232 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 232 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 232 or a functional fragment thereof.

In alternate embodiments, pbt and buk are replaced with TesB (SEQ ID NO: 15)

In some embodiments, the butyrate cassette is driven by an inducible promoter. For example, other FNR promoters can be used in lieu of ydfZ, e.g., in SEQ ID NO: 180-196.

Non-limiting FNR promoter sequences are provided herein. In some embodiments, the genetically engineered bacteria of the invention comprise a butyrate cassette under the control of one or more of promoter sequences found in Table 6, e.g., nirB promoter, ydfZ promoter, nirB promoter fused to a strong ribosome binding site, ydfZ promoter fused to a strong ribosome binding site, fnrS, an anaerobically induced small RNA gene (fnrS promoter), nirB promoter fused to a crp binding site, and fnrS fused to a crp binding site.

In some embodiments, the butyrate cassette is under the control of a promoter which is inducible by metabolites present in the gut. In some embodiments, the butyrate cassette is induced by HE-specific molecules or metabolites indicative of liver damage, e.g., bilirubin. In some embodiments, the butyrate cassette is placed under the control of promoter, which is inducible by inflammation or an inflammatory response (e.g., RNS or ROS promoter).

In some embodiments, the genetically engineered bacteria comprise a butyrate cassette driven by a promoter induced by a molecule or metabolite. Promoters that respond to one of these molecules or their metabolites may be used in the genetically engineered bacteria provided herein.

In some embodiments, the butyrate cassette is inducible by arabinose and is driven by the AraBAD promoter.

Example 17 Comparison of In Vitro Butyrate Production Efficacy of Chromosomal Insertion and Plasmid-Bearing Engineered Bacterial Strains

The in vitro butyrate production efficacy of engineered bacterial strains harboring a chromosomal insertion of a butyrate cassette was compared to a strain bearing a butyrate cassette on a plasmid. SYN1001 and SYN1002 harbor a chromosomal insertion between the agal/rsml locus of a butyrate cassette (either ter→tesB or ter→pbt-buk, respectively) driven by an fnr inducible promoter. These strains were compared side by side with the low copy plasmid strain SYN501 (Logic156 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid) also driven by an fnr inducible promoter. Butyrate levels in the media were measured at 4 and 24 hours post anaerobic induction.

Briefly, 3 ml LB was inoculated with bacteria from frozen glycerol stocks. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 dilution into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10⁸ CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At indicated times (4 and 24 h), 120 ul cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate concentrations (as described in Example 21). Results are depicted in FIG. 11, and show that SYN1001 and SYN1002 give comparable butyrate production to the plasmid strain SYN501.

TABLE 40 FRNRs Butyrate Cassette Sequences Description Sequence Pfnrs-ter-thiA1-hbd-ctr2- GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTAT tesB GGTTGCATCGTAGTAAATGGTTGTAACAAAAGC SEQ ID NO: 233, e.g. AATTTTTCCGGCTGTCTGTATACAAAAACGCCGC integrated into the AAAGTTTGAGCGAAGTCAATAAACTCTCTACCC chromosome in ATTCAGGGCAATATCTCTCTTGGATCCAAAGTGA SYN1001 Pfnrs: ACTCTAGAAATAATTTTGTTTAACTTTAAGAAGG uppercase; butyrate AGATATACATatgatcgtaaaacctatggtacgcaacaatatctgcctg cassette: lower case aacgcccatcctcagggctgcaagaagggagtggaagatcagattgaatatac caagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaac gttctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctg cgttcggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcag aaaccaaatatggtacaccgggatggtacaataatttggcatttgatgaagcggc aaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttcagacga gatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatct gatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatcatg cacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatc cgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaa gcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaag cagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagtt atattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaagg ccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtc aatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccg taatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaag ggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcctgt accgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgat tgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagt cacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgattt cttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaac gcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgc cagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttca gcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacat aactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttg gacaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaa ccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatggcat ctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaaca tgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgat gctgcttttgttgattcaatgataaaagatggattatcagacatatttaataactatca catgggtattactgctgaaaacatagcagagcaatggaatataactagagaaga acaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaa ggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgaca ctgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgct aagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcag gaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaaga actaggaatagagcctcagcaactatagtacttatggaacagctggtgagacc ctaaaataatgggatatggaccagaccagcaactaaaaaagcatagaagctgc taatatgactattgaagatatagatttagagaagctaatgaggcatagctgccca atctgtagctgtaataagagacttaaatatagatatgaataaagttaatgaaatggt ggagcaatagctataggacatccaataggatgctcaggagcaagaatacttact acacattatatgaaatgaagagaagagatgctaaaactggtcagctacactagt ataggcggtggaatgggaactacataatagttaagagatagtaagaaggagat atacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtaca aacattgcaagagtggacatgatgtatgataaagagtagaactcaaggtgctat agataaatgatagcatattagataaaaatttaactaagttagttactaagggaaaa atggatgaagctacaaaagcagaaatattaagtcatgaagacaactactaattat gaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatat aaagaaagatgattcaagttactagatgaattatgtaaagaagatactatcaggc aacaaatacttcatcattatctataacagaaatagatcactactaagcgcccaga taaagttataggaatgcatttattaatccagacctatgatgaaattagagaagtta taagtggtcagttaacatcaaaagttacattgatacagtatttgaattatctaagagt atcaataaagtaccagtagatgtatctgaatctcctggatagtagtaaatagaata cttatacctatgataaatgaagctgaggtatatatgcagatggtgagcaagtaaa gaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccacta gcattaggtgatttaatcggattagatgagattagctataatgaacgattatatact gaataggagatactaaatatagacctcatccactatagctaaaatggttagagct aatcaattaggaagaaaaactaagataggattctatgattataataaataataaga aggagatatacatatgagtacaagtgatgaaaagatatgagaatgtagctgaga agtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgca ataaattcaaagacatagaagaacatatgaagtatagtagatattaataatgatg aaactattgatgagtaatattgacaggggaaggaaaggcatagtagctggagc agatattgcatacatgaaagatttagatgctgtagctgctaaagatatagtatctta ggagcaaaagataggagaaatagaaaatagtaaaaaagtagtgatagctgct gtaaacggatttgattaggtggaggatgtgaacttgcaatggcatgtgatataag aattgcatctgctaaagctaaataggtcagccagaagtaactcaggaataactc caggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagca aaagaattaatattacaggtcaagttataaaagctgatgaagctgaaaaaatagg gctagtaaatagagtcgagagccagacatataatagaagaagagagaaattag ctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaata caacaggtgctcaaactgatataaatactggaatagatatagaatctaatttatag gtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaaga gagaagctaacatataaaagggtaataagaaggagatatacatatgagtcagg cgctaaaaaatttactgacattgaaaatctggaaaaaattgaggaaggactattc gcggccagagtgaagatttaggtttacgccaggtgtttggcggccaggtcgtgg gtcaggccagtatgctgcaaaagagaccgtccctgaagagcggctggtacatt cgatcacagctactacttcgccctggcgatagtaagaagccgattatttatgatgt cgaaacgctgcgtgacggtaacagcttcagcgcccgccgggagctgctattca aaacggcaaaccgattattatatgactgcctctaccaggcaccagaagcggga tcgaacatcaaaaaacaatgccgtccgcgccagcgcctgatggcctccatcgg aaacgcaaatcgcccaatcgctggcgcacctgctgccgccagtgctgaaagat aaattcatctgcgatcgtccgctggaagtccgtccggtggagatcataacccact gaaaggtcacgtcgcagaaccacatcgtcaggtgtggatccgcgcaaatggta gcgtgccggatgacctgcgcgttcatcagtatctgctcggttacgcttctgatctta acttcctgccggtagctctacagccgcacggcatcggttttctcgaaccggggat tcagattgccaccattgaccattccatgtggttccatcgcccgtttaatttgaatgaa tggctgctgtatagcgtggagagcacctcggcgtccagcgcacgtggctttgtg cgcggtgagttttatacccaagacggcgtactggttgcctcgaccgttcaggaa ggggtgatgcgtaatcacaattaa Pfnrs-ter-thiA1-hbd-crt2- GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTAT pbt-buk GGTTGCATCGTAGTAAATGGTTGTAACAAAAGC (SEQ ID NO: 234), e.g. AATTTTTCCGGCTGTCTGTATACAAAAACGCCGC integrated into the AAAGTTTGAGCGAAGTCAATAAACTCTCTACCC chromosome in ATTCAGGGCAATATCTCTCTTGGATCCAAAGTGA SYN1002 Pfnrs: ACTCTAGAAATAATTTTGTTTAACTTTAAGAAGG uppercase; butyrate AGATATACATatgatcgtaaaacctatggtacgcaacaatatctgcctg cassette: lower case aacgcccatcctcagggctgcaagaagggagtggaagatcagattgaatatac caagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaac gttctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctg cgttcggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcag aaaccaaatatggtacaccgggatggtacaataatttggcatttgatgaagcggc aaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttcagacga gatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatct gatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatcatg cacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatc cgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaa gcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaag cagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagtt atattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaagg ccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtc aatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccg taatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaag ggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcctgt accgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgat tgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagt cacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgattt cttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaac gcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgc cagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttca gcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacat aactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttg gacaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaa ccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatggcat ctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaaca tgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgat gctgcttttgttgattcaatgataaaagatggattatcagacatatttaataactatca catgggtattactgctgaaaacatagcagagcaatggaatataactagagaaga acaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaa ggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgaca ctgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgct aagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcag gaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaaga actaggaatagagcctcagcaactatagtacttatggaacagctggtgagacc ctaaaataatgggatatggaccagaccagcaactaaaaaagcatagaagctgc taatatgactattgaagatatagatttagagaagctaatgaggcatagctgccca atctgtagctgtaataagagacttaaatatagatatgaataaagttaatgaaatggt ggagcaatagctataggacatccaataggatgctcaggagcaagaatacttact acacattatatgaaatgaagagaagagatgctaaaactggtcagctacactagt ataggcggtggaatgggaactacataatagttaagagatagtaagaaggagat atacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtaca aacattgcaagagtggacatgatgtatgataaagagtagaactcaaggtgctat agataaatgatagcatattagataaaaatttaactaagttagttactaagggaaaa atggatgaagctacaaaagcagaaatattaagtcatgaagacaactactaattat gaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatat aaagaaagatgattcaagttactagatgaattatgtaaagaagatactatcaggc aacaaatacttcatcattatctataacagaaatagatcactactaagcgcccaga taaagttataggaatgcatttattaatccagacctatgatgaaattagagaagtta taagtggtcagttaacatcaaaagttacattgatacagtatttgaattatctaagagt atcaataaagtaccagtagatgtatctgaatctcctggatagtagtaaatagaata cttatacctatgataaatgaagctgaggtatatatgcagatggtgagcaagtaaa gaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccacta gcattaggtgatttaatcggattagatgagattagctataatgaacgattatatact gaataggagatactaaatatagacctcatccactatagctaaaatggttagagct aatcaattaggaagaaaaactaagataggattctatgattataataaataataaga aggagatatacatatgagtacaagtgatgaaaagatatgagaatgtagctgaga agtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgca ataaattcaaagacatagaagaacatatgaagtatagtagatattaataatgatg aaactattgatgagtaatattgacaggggaaggaaaggcatagtagctggagc agatattgcatacatgaaagatttagatgctgtagctgctaaagatatagtatctta ggagcaaaagataggagaaatagaaaatagtaaaaaagtagtgatagctgct gtaaacggatttgattaggtggaggatgtgaacttgcaatggcatgtgatataag aattgcatctgctaaagctaaataggtcagccagaagtaactcaggaataactc caggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagca aaagaattaatattacaggtcaagttataaaagctgatgaagctgaaaaaatagg gctagtaaatagagtcgagagccagacatataatagaagaagagagaaattag ctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaata caacaggtgctcaaactgatataaatactggaatagatatagaatctaatttatag gtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaaga gagaagctaacatataaaagggtaataagaaggagatatacatatgagaagatt gaagaagtaattaagtagcaaaagaaagaggacctaaaactatatcagtagcat gagccaagataaagaagattaatggcagagaaatggctagaaaagaaaaaat agcaaatgccatatagtaggagatatagaaaagactaaagaaattgcaaaaag catagacatggatatcgaaaattatgaactgatagatataaaagatttagcagaag catctctaaaatctgagaattagatcacaaggaaaagccgacatggtaatgaaa ggcttagtagacacatcaataatactaaaagcagattaaataaagaagtaggtct tagaactggaaatgtattaagtcacgtagcagtatttgatgtagagggatatgata gattattatcgtaactgacgcagctatgaacttagctcctgatacaaatactaaaa agcaaatcatagaaaatgatgcacagtagcacattcattagatataagtgaacc aaaagagctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagatac agagaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaaggag tatggaggtgggccattgcaattgataatgcagtatattagaagcagctaaaca taaaggtataaatcatcctgtagcaggacgagctgatatattattagccccagata ttgaaggtggtaacatattatataaagctaggtattcactcaaaatcaaaaaatgc aggagttatagaggggctaaagcaccaataatattaacactagagcagacagt gaagaaactaaactaaactcaatagcataggtgattaatggcagcaaaggcata ataagaaggagatatacatatgagcaaaatatttaaaatcttaacaataaatcctg gacgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaac ataagacattatcagaagaaataggaaaatatgagaaggtgtctgaccaatag aatacgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacat ctgaattagatgctgtagtaggtagaggaggacacttaaacctataaaaggtggt acttattcagtaagtgctgctatgattgaagatttaaaagtgggagattaggagaa cacgcacaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaat gaccacatacatagtagaccctgagagtagatgaattagaagatgagctagaa tactggtatgcctgaaataagtagagcaagtgtagtacatgattaaatcaaaag gcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatataaat cttatagagcacacatgggtggaggagtactgaggagctcataaaaatggtaa aatagtagatgttgcaaacgcattagatggagaaggacctttctctccagaaaga agtggtggactaccagtaggtgcattagtaaaaatgtgattagtggaaaatatac tcaagatgaaattaaaaagaaaataaaaggtaatggcggactagagcatactta aacactaatgatgctagagaagagaagaaagaattgaagctggtgatgaaaaa gctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagctag tgctgcagacttaagggagatgtaaaagcaatattattaactggtggaatcgcat attcaaaaatgatacagaaatgattgcagatagagttaaatttatagcagatgtaa aagatatccaggtgaagatgaaatgattgcattagctcaaggtggacttagagat taactggtgaagaagaggctcaagtttatgataactaa PfNRS (ribosome GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTAT binding site is GGTTGCATCGTAGTAAATGGTTGTAACAAAAGC underlined) AATTTTTCCGGCTGTCTGTATACAAAAACGCCGC (SEQ ID NO: 235) AAAGTTTGAGCGAAGTCAATAAACTCTCTACCC ATTCAGGGCAATATCTCTCTTGGATCCAAAGTGA ACTCTAGAAATAATTTTGTTTAACTTTAAGAAGG AGATATACAT Ribosome binding site CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA and leader region (SEQ GATATACAT ID NO: 236)

Example 18 Assessment of Intestinal Butyrate Levels In Response to SYN501 Administration In Mice

To determine efficacy of butyrate production by the genetically engineered bacteria in vivo, the levels of butyrate upon administration of SYN501 (Logic156 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid)) to C57BL6 mice was first assessed in the feces. Water containing 100 mM butyrate was used as a control.

On day 1, C57BL6 mice (24 total animals) were weighed and randomized into 4 groups; Group 1: H2O2O control (n=6); Group 2-100 mM butyrate (n=6); Group 3-streptomycin resistant Nissle (n=6); Group 4-SYN501 (n=6). Mice were either gavaged with 100 ul streptomycin resistant Nissle or SYN501, and group 2 was changed to H20(+)100 mM butyrate at a dose of 10e10 cells/100u1. On days 2-4, mice were weighted and Groups 3 and 4 were gavaged in the AM and the PM with streptomycin resistant Nissle or SYN501. On day 5, mice were weighed and Groups 3 and 4 were gavaged in the am with streptomycin resistant Nissle or SYN501, and feces was collected and butyrate concentrations determined as described in Example 23. Results are depicted in FIG. 10. Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H20 (+) 200 mM butyrate.

Next the effects of SYN501 on levels of butyrate in the cecum, cecal effluent, large intestine, and large intestine effluent are assessed. Because baseline concentrations of butyrate are high in these compartments, an antibiotic treatment is administered in advance to clear out the bacteria responsible for butyrate production in the intestine. As a result, smaller differences in butyrate levels can be more accurately observed and measured. Water containing 100 mM butyrate is used as a control.

During week 1 of the study, animals are treated with an antibiotic cocktail in the drinking water to reduce the baseline levels of resident microflora. The antibiotic cocktail is composed of ABX-ampicillin, vancomycin, neomycin, and metronidazole. During week 2 animals are orally administered 100 ul of streptomycin resistant Nissle or engineered strain SYN501 twice a day for five days (at a dose of 10e10 cells/100 ul).

On day 1, C57BL6 (Female, 8 weeks) are separated into four groups as follows: Group 1: H20 control (n=10); Group 2: 100 mM butyrate (n=10); Group 3: streptomycin resistant Nissle (n=10); Group 4: SYN501 (n=10). Animals are weighed and feces is collected from the animals (T=0-time point). Animals are changed to H2O (+) antibiotic cocktail. On day 5, animals are weighed and feces is collected (time point T=5d). The H2O (+) antibiotic cocktail bottles are changed. On day 8, the mice are weighed and feces is collected. Mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. The water in all cages is changed to water without antibiotic. Group 2 is provided with 100 mM butyrate in H2O. On days 9-11, mice are weighed, and mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. On day 12, mice are gavaged with streptomycin resistant Nissle or SYN501 in the AM, and 4 hours post dose, blood is harvested, and cecal and large intestinal contents, and tissue, and feces are collected and processed for analysis.

Example 19 Measurement of Satiety Markers Upon Administration of SYN501 In Vivo

To determine whether administration of a butyrate producing strain might result in increased levels of satiety markers, SYN501 is administered to 10-week old C57BL6 (10 weeks) and blood levels of GLP1 and insulin are measured. Butyrate in H20 at 100 mM is used as a control (e.g., as described in Lin et al., Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms, PLOS One, April 2012 | Volume 7 ═ Issue 4 ═ e35240).

On day 1, animals are randomized and distributed into 5 groups as follows: Group 1: Time 0 control (n=6); Group 2-H20 (+) 100 mM butyrate, 10 min (n=6); Group 3-SYN501, 30 min (n=6); Group 4-SYN501, 4h (n=6); Group 5-H20 (+) 100 mM butyrate, 4h (n=6). Mice are fasted overnight. On day 2, mice are gavaged with either H20(+)100 mM butyrate or SYN501. Then, blood is harvested via cardiac bleed at the following time points post dose: Group 1 is Time 0; Group 2 (H20 (+) 100 mM butyrate) at 10 min; Group 3 (SYN501) at 30 min; Group 4 (SYN501) at 4 h; Group 5 (H20 (+) 100 mM butyrate) at 4 h. Serum is analyzed by ELISA for GLP-1 and insulin. Fecal samples are analyzed for butyrate by MS as described herein.

Example 20 Comparison of Butyrate Production Levels Between the Genetically Engineered Bacteria Encoding a Butyrate Cassette and Selected Clostridia strains

The efficacy of butyrate production in SYN501 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid) was compared to CBM588 (Clostridia butyricum MIYARISAN, a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain).

Briefly, overnight cultures of SYN501 were diluted 1:100 dilution and was grown in RCM (Reinforced Clostridial Media, which is similar to LB but contains 05% glucose) at 37 C with shaking for 2 hours, then either moved into the anaerobic chamber or left aerobically shaking. Clostridial strains were only grown anaerobically.

At indicated times (2, 8, 24, and 48 h), 120 ul cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate concentrations (as described in Example 21). Results are depicted in FIG. 12, and show that SYN501 produces butyrate levels comparable to Clostridium spp. in RCM media

Example 21 Quantification of Butyrate by LC-MS/NIS

To obtain the butyrate measurements in Example 37 a LC-MS/MS protocol for butyrate quantification was used.

Sample Preparation

First, fresh 1000, 500, 250, 100, 20, 4 and 0.8 μm/mL sodium butyrate standards were prepared in water. Then, 10 μL of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90 μL of 67% ACN (60 uL ACN+30 uL water per reaction) with 4 ug/mL of butyrate-d7 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000 rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 20 μL of diluted samples were added to 180 μL of a buffer containing 10 mM MES pH4.5, 20 mM EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), and 20 mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour.

LC-MS/MS Method

Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 41 and Table 42. Tandem Mass Spectrometry details are found in Table 43.

TABLE 41 HPLC Details Column Thermo Aquasil C18 column, 5 μm (50 × 2.1 mm) Mobile 100% H2O, 0.1% Formic Phase A Acid Mobile 100% ACN, 0.1% Formic Phase B Acid Injection 10 uL volume

TABLE 42 HPLC Method Total Flow Time Rate (min) (μL/min) A % B % 0 0.5 100 0 1 0.5 100 0 2 0.5 10 90 4 0.5 10 90 4.01 0.5 100 0 4.25 0.5 100 0

TABLE 43 Tandem Mass Spectrometry Details Ion Source HESI-II Polarity Positive SRM Butyrate 170.0/71.1, transitions Butyrate d7 177.1/78.3

Example 22 Quantification of Butyrate in Feces by LC-MS/MS Sample Preparation

Fresh 1000, 500, 250, 100, 20, 4 and 0.8m/mL sodium butyrate standards were prepared in water. Single fecal pellets were ground in 100 uL water and centrifuged at 15,000 rpm for 5min at 4° C. 10 μL of the sample (fecal supernatant and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90pL of the derivatizing solution containing 50 mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphospine in acetonitrile with 5 ug/mL of butyrate-d₇ were added to each sample. The plate was heat-sealed and incubated at 60° C. for 1 hr. The plate was then centrifuged at 4,000 rpm for 5min and 20 μL of the derivatized samples mixed to 180 μL of 22% acetonitrile with 0.1% formic acid.

LC-MS/MS Method

Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 44 and Table 45. Tandem Mass Spectrometry details are found in Table 46.

TABLE 44 HPLC Details Column Luna phenomenex C18 column, 5 μm (100 × 2.1 mm) Mobile 100% H2O, 0.1% Formic Phase A Acid Mobile 100% ACN, 0.1% Formic Phase B Acid Injection 10 uL volume

TABLE 45 HPLC Method Total Flow Time Rate (min) (μL/min) A % B % 0 0.5 95 5 0.5 0.5 95 5 1.5 0.5 10 90 3.5 0.5 10 90 3.51 0.5 95 5 3.75 0.5 95 5

TABLE 46 Tandem Mass Spectrometry Details Ion Source HESI-II Polarity Positive SRM Butyrate 230.1/143.1, transitions Butyrate d7 237.1/143.1

Example 23 Production of Propionate Through the Sleeping Beauty Mutase Pathway in Genetically Engineered E. coli BW25113 and Nissle

In E. coli , a four gene operon, sbm-ygfD-ygfG-ygfH (sleeping beauty mutase pathway) has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro. While the sleeping beauty mutase pathway is present in E. coli , it is not under the control of a strong promoter and has shown low activity in vivo.

The utility of this operon for the production of propionate was assessed. Because E. coli Nissle does not have the complete operon, initial experiments were conducted in E. coli K12 (BW25113).

First, the native promoter for the sleeping beauty mutase operon on the chromosome in the BW25113 strain was replaced with a fnr promoter (BW25113 ldhA::frt; PfnrS-SBM-cam). The sequence for this construct is provided in Table 47. Mutation of the lactate dehydrogenase gene (ldhA) reportedly increases propionate production, and this mutation is therefore also added in certain embodiments.

TABLE 47 SBM Construct Sequences Description Sequence BW25113 fnrS SBM

construct (BW25113 frt-

cam-frt-PfnrS-sbm, ygfD,

CCGCCGGG ygfG, ygfH), comprising rrnB AGCGGATTTGAACGTTGCGAAGCAACGGC terminator 1, rrnB terminator 2 CCGGAGGGTGGCGGGCAGGACGCCCGCC (both italic, uppercase), cat ATAAACTGCCAGGCATCAAATTAAGC

promoter and cam resistance

TGCGTGG gene (encoded on the CCAGTGCCAAGCTTGCATGCAGATTGCAG lagging strand underlined CATTACACGTCTTGAGCGATTGTGTAGGCT uppercase), frt sites (italic GGAGCTGCTTC

underlined), FNRS promoter

ATTT bold lowercase, with RBS and AAATGGCGCGCCTTACGCCCCGCCCTGCC leader region bold and A CTCATCGCAGTACTGTTGTATTCATTAAG underlined and FNR binding site CATCTGCCGACATGGAAGCCATCACAAAC in bold and italics); sleeping GGCATGATGAACCTGAATCGCCAGCGGCA beauty operon (sbm, ygfD, TCAGCACCTTGTCGCCTTGCGTATAATATT ygfG, ygfH) bold and uppercase TGCCCATGGTGAAAACGGGGGCGAAGAAG (SEQ ID NO: 237) TTGTCCATATTGGCCACGTTTAAATCAAAA CTGGTGAAACTCACCCAGGGATTGGCTGA GACGAAAAACATATTCTCAATAAACCCTTT AGGGAAATAGGCCAGGTTTTCACCGTAAC ACGCCACATCTTGCGAATATATGTGTAGAA ACTGCCGGAAATCGTCGTGGTATTCACTC CAGAGCGATGAAAACGTTTCAGTTTGCTC ATGGAAAACGGTGTAACAAGGGTGAACAC TATCCCATATCACCAGCTCACCGTCTTTCA TTGCCATACGTAATTCCGGATGAGCATTCA TCAGGCGGGCAAGAATGTGAATAAAGGCC GGATAAAACTTGTGCTTATTTTTCTTTACG GTCTTTAAAAAGGCCGTAATATCCAGCTGA ACGGTCTGGTTATAGGTACATTGAGCAAC TGACTGAAATGCCTCAAAATGTTCTTTACG ATGCCATTGGGATATATCAACGGTGGTAT ATCCAGTGATTTTTTTCTCCATTTTAGCTT CCTTAGCTCCTGAAAATCTCGACAACTCAA AAAATACGCCCGGTAGTGATCTTATTTCAT TATGGTGAAAGTTGGAACCTCTTACGTGC CGATCA ACGTCTCATTTTCGCCAAAAGTTG GCCCAGGGCTTCCCGGTATCAACAGGGAC ACCAGGATTTATTTATTCTGCGAAGTGATC TTCCGTCACAGGTAGGCGCGCC

GGAATA GGAACTAAGGAGGATATTCATATGGACCA TGGCTAATTCCCAGGTACCagttgttcttattggtggt gttgctttatggttgcatcgtagtaaatggttgtaacaaaagcaattttt ccggctgtctgtatacaaaaacgccgcaaagt

ta aactctctacccattcagggcaatatctctcttggatccaaagtgaa ct ctagaaataattttgtttaactttaagaaggagatatacat ATGTC TAACGTGCAGGAGTGGCAACAGCTTGCCA ACAAGGAATTGAGCCGTCGGGAGAAAACT GTCGACTCGCTGGTTCATCAAACCGCGGA AGGGATCGCCATCAAGCCGCTGTATACCG AAGCCGATCTCGATAATCTGGAGGTGACA GGTACCCTTCCTGGTTTGCCGCCCTACGTT CGTGGCCCGCGTGCCACTATGTATACCGC CCAACCGTGGACCATCCGTCAGTATGCTG GTTTTTCAACAGCAAAAGAGTCCAACGCTT TTTATCGCCGTAACCTGGCCGCCGGGCAA AAAGGTCTTTCCGTTGCGTTTGACCTTGCC ACCCACCGTGGCTACGACTCCGATAACCC GCGCGTGGCGGGCGACGTCGGCAAAGCG GGCGTCGCTATCGACACCGTGGAAGATAT GAAAGTCCTGTTCGACCAGATCCCGCTGG ATAAAATGTCGGTTTCGATGACCATGAATG GCGCAGTGCTACCAGTACTGGCGTTTTAT ATCGTCGCCGCAGAAGAGCAAGGTGTTAC ACCTGATAAACTGACCGGCACCATTCAAA ACGATATTCTCAAAGAGTACCTCTGCCGCA ACACCTATATTTACCCACCAAAACCGTCAA TGCGCATTATCGCCGACATCATCGCCTGG TGTTCCGGCAACATGCCGCGATTTAATACC ATCAGTATCAGCGGTTACCACATGGGTGA AGCGGGTGCCAACTGCGTGCAGCAGGTAG CATTTACGCTCGCTGATGGGATTGAGTAC ATCAAAGCAGCAATCTCTGCCGGACTGAA AATTGATGACTTCGCTCCTCGCCTGTCGTT CTTCTTCGGCATCGGCATGGATCTGTTTAT GAACGTCGCCATGTTGCGTGCGGCACGTT ATTTATGGAGCGAAGCGGTCAGTGGATTT GGCGCACAGGACCCGAAATCACTGGCGCT GCGTACCCACTGCCAGACCTCAGGCTGGA GCCTGACTGAACAGGATCCGTATAACAAC GTTATCCGCACCACCATTGAAGCGCTGGC TGCGACGCTGGGCGGTACTCAGTCACTGC ATACCAACGCCTTTGACGAAGCGCTTGGT TTGCCTACCGATTTCTCAGCACGCATTGCC CGCAACACCCAGATCATCATCCAGGAAGA ATCAGAACTCTGCCGCACCGTCGATCCAC TGGCCGGATCCTATTACATTGAGTCGCTG ACCGATCAAATCGTCAAACAAGCCAGAGC TATTATCCAACAGATCGACGAAGCCGGTG GCATGGCGAAAGCGATCGAAGCAGGTCTG CCAAAACGAATGATCGAAGAGGCCTCAGC GCGCGAACAGTCGCTGATCGACCAGGGCA AGCGTGTCATCGTTGGTGTCAACAAGTAC AAACTGGATCACGAAGACGAAACCGATGT ACTTGAGATCGACAACGTGATGGTGCGTA ACGAGCAAATTGCTTCGCTGGAACGCATT CGCGCCACCCGTGATGATGCCGCCGTAAC CGCCGCGTTGAACGCCCTGACTCACGCCG CACAGCATAACGAAAACCTGCTGGCTGCC GCTGTTAATGCCGCTCGCGTTCGCGCCAC CCTGGGTGAAATTTCCGATGCGCTGGAAG TCGCTTTCGACCGTTATCTGGTGCCAAGCC AGTGTGTTACCGGCGTGATTGCGCAAAGC TATCATCAGTCTGAGAAATCGGCCTCCGA GTTCGATGCCATTGTTGCGCAAACGGAGC AGTTCCTTGCCGACAATGGTCGTCGCCCG CGCATTCTGATCGCTAAGATGGGCCAGGA TGGACACGATCGCGGCGCGAAAGTGATCG CCAGCGCCTATTCCGATCTCGGTTTCGAC GTAGATTTAAGCCCGATGTTCTCTACACCT GAAGAGATCGCCCGCCTGGCCGTAGAAAA CGACGTTCACGTAGTGGGCGCATCCTCAC TGGCTGCCGGTCATAAAACGCTGATCCCG GAACTGGTCGAAGCGCTGAAAAAATGGGG ACGCGAAGATATCTGCGTGGTCGCGGGTG GCGTCATTCCGCCGCAGGATTACGCCTTC CTGCAAGAGCGCGGCGTGGCGGCGATTTA TGGTCCAGGTACACCTATGCTCGACAGTG TGCGCGACGTACTGAATCTGATAAGCCAG CATCATGATTAATGAAGCCACGCTGGCAG AAAGTATTCGCCGCTTACGTCAGGGTGAG CGTGCCACACTCGCCCAGGCCATGACGCT GGTGGAAAGCCGTCACCCGCGTCATCAGG CACTAAGTACGCAGCTGCTTGATGCCATTA TGCCGTACTGCGGTAACACCCTGCGACTG GGCGTTACCGGCACCCCCGGCGCGGGGAA AAGTACCTTTCTTGAGGCCTTTGGCATGTT GTTGATTCGAGAGGGATTAAAGGTCGCGG TTATTGCGGTCGATCCCAGCAGCCCGGTC ACTGGCGGTAGCATTCTCGGGGATAAAAC CCGCATGAATGACCTGGCGCGTGCCGAAG CGGCGTTTATTCGCCCGGTACCATCCTCC GGTCATCTGGGCGGTGCCAGTCAGCGAGC GCGGGAATTAATGCTGTTATGCGAAGCAG CGGGTTATGACGTAGTGATTGTCGAAACG GTTGGCGTCGGGCAGTCGGAAACAGAAGT CGCCCGCATGGTGGACTGTTTTATCTCGTT GCAAATTGCCGGTGGCGGCGATGATCTGC AGGGCATTAAAAAAGGGCTGATGGAAGTG GCTGATCTGATCGTTATCAACAAAGACGAT GGCGATAACCATACCAATGTCGCCATTGC CCGGCATATGTACGAGAGTGCCCTGCATA TTCTGCGACGTAAATACGACGAATGGCAG CCACGGGTTCTGACTTGTAGCGCACTGGA AAAACGTGGAATCGATGAGATCTGGCACG CCATCATCGACTTCAAAACCGCGCTAACTG CCAGTGGTCGTTTACAACAAGTGCGGCAA CAACAATCGGTGGAATGGCTGCGTAAGCA GACCGAAGAAGAAGTACTGAATCACCTGT TCGCGAATGAAGATTTCGATCGCTATTACC GCCAGACGCTTTTAGCGGTCAAAAACAAT ACGCTCTCACCGCGCACCGGCCTGCGGCA GCTCAGTGAATTTATCCAGACGCAATATTT TGATTAAAGGAATTTTTATGTCTTATCAGTA TGTTAACGTTGTCACTATCAACAAAGTGGC GGTCATTGAGTTTAACTATGGCCGAAAACT TAATGCCTTAAGTAAAGTCTTTATTGATGA TCTTATGCAGGCGTTAAGCGATCTCAACC GGCCGGAAATTCGCTGTATCATTTTGCGC GCACCGAGTGGATCCAAAGTCTTCTCCGC AGGTCACGATATTCACGAACTGCCGTCTG GCGGTCGCGATCCGCTCTCCTATGATGAT CCATTGCGTCAAATCACCCGCATGATCCAA AAATTCCCGAAACCGATCATTTCGATGGTG GAAGGTAGTGTTTGGGGTGGCGCATTTGA AATGATCATGAGTTCCGATCTGATCATCGC CGCCAGTACCTCAACCTTCTCAATGACGCC TGTAAACCTCGGCGTCCCGTATAACCTGG TCGGCATTCACAACCTGACCCGCGACGCG GGCTTCCACATTGTCAAAGAGCTGATTTTT ACCGCTTCGCCAATCACCGCCCAGCGCGC GCTGGCTGTCGGCATCCTCAACCATGTTG TGGAAGTGGAAGAACTGGAAGATTTCACC TTACAAATGGCGCACCACATCTCTGAGAA AGCGCCGTTAGCCATTGCCGTTATCAAAG AAGAGCTGCGTGTACTGGGCGAAGCACAC ACCATGAACTCCGATGAATTTGAACGTATT CAGGGGATGCGCCGCGCGGTGTATGACAG CGAAGATTACCAGGAAGGGATGAACGCTT TCCTCGAAAAACGTAAACCTAATTTCGTTG GTCATTAATCCCTGCGAACGAAGGAGTAAA AATGGAAACTCAGTGGACAAGGATGACCG CCAATGAAGCGGCAGAAATTATCCAGCAT AACGACATGGTGGCATTTAGCGGCTTTAC CCCGGCGGGTTCGCCGAAAGCCCTACCCA CCGCGATTGCCCGCAGAGCTAACGAACAG CATGAGGCCAAAAAGCCGTATCAAATTCG CCTTCTGACGGGTGCGTCAATCAGCGCCG CCGCTGACGATGTACTTTCTGACGCCGAT GCTGTTTCCTGGCGTGCGCCATATCAAAC ATCGTCCGGTTTACGTAAAAAGATCAATCA GGGCGCGGTGAGTTTCGTTGACCTGCATT TGAGCGAAGTGGCGCAAATGGTCAATTAC GGTTTCTTCGGCGACATTGATGTTGCCGTC ATTGAAGCATCGGCACTGGCACCGGATGG TCGAGTCTGGTTAACCAGCGGGATCGGTA ATGCGCCGACCTGGCTGCTGCGGGCGAAG AAAGTGATCATTGAACTCAATCACTATCAC GATCCGCGCGTTGCAGAACTGGCGGATAT TGTGATTCCTGGCGCGCCACCGCGGCGCA ATAGCGTGTCGATCTTCCATGCAATGGATC GCGTCGGTACCCGCTATGTGCAAATCGAT CCGAAAAAGATTGTCGCCGTCGTGGAAAC CAACTTGCCCGACGCCGGTAATATGCTGG ATAAGCAAAATCCCATGTGCCAGCAGATT GCCGATAACGTGGTCACGTTCTTATTGCA GGAAATGGCGCATGGGCGTATTCCGCCGG AATTTCTGCCGCTGCAAAGTGGCGTGGGC AATATCAATAATGCGGTAATGGCGCGTCT GGGGGAAAACCCGGTAATTCCTCCGTTTA TGATGTATTCGGAAGTGCTACAGGAATCG GTGGTGCATTTACTGGAAACCGGCAAAAT CAGCGGGGCCAGCGCCTCCAGCCTGACAA TCTCGGCCGATTCCCTGCGCAAGATTTAC GACAATATGGATTACTTTGCCAGCCGCATT GTGTTGCGTCCGCAGGAGATTTCCAATAA CCCGGAAATCATCCGTCGTCTGGGCGTCA TCGCTCTGAACGTCGGCCTGGAGTTTGAT ATTTACGGGCATGCCAACTCAACACACGT AGCCGGGGTCGATCTGATGAACGGCATCG GCGGCAGCGGTGATTTTGAACGCAACGCG TATCTGTCGATCTTTATGGCCCCGTCGATT GCTAAAGAAGGCAAGATCTCAACCGTCGT GCCAATGTGCAGCCATGTTGATCACAGCG AACACAGCGTCAAAGTGATCATCACCGAA CAAGGGATCGCCGATCTGCGCGGTCTTTC CCCGCTTCAACGCGCCCGCACTATCATTG ATAATTGTGCACATCCTATGTATCGGGATT ATCTGCATCGCTATCTGGAAAATGCGCCT GGCGGACATATTCACCACGATCTTAGCCA CGTCTTCGACTTACACCGTAATTTAATTGC AACCGGCTCGATGCTGGGTTAA FNRS promoter bold lowercase, agttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggtt with RBS and leader region gtaacaaaagcaatttttccggctgtctgtatacaaaaacgccgcaaa bold and underlined, and FNR gt

taaactctctacccattcagggcaatatctctct binding site bold and italics); tggatccaaagtgaa ctctagaaataattttgtttaactttaagaagga sleeping beauty operon (sbm, gatatacat ATGTCTAACGTGCAGGAGTGGCAA ygfD, ygfG, ygfH) bold and CAGCTTGCCAACAAGGAATTGAGCCGTCG uppercase GGAGAAAACTGTCGACTCGCTGGTTCATC (SEQ ID NO: 238) AAACCGCGGAAGGGATCGCCATCAAGCCG CTGTATACCGAAGCCGATCTCGATAATCTG GAGGTGACAGGTACCCTTCCTGGTTTGCC GCCCTACGTTCGTGGCCCGCGTGCCACTA TGTATACCGCCCAACCGTGGACCATCCGT CAGTATGCTGGTTTTTCAACAGCAAAAGA GTCCAACGCTTTTTATCGCCGTAACCTGGC CGCCGGGCAAAAAGGTCTTTCCGTTGCGT TTGACCTTGCCACCCACCGTGGCTACGAC TCCGATAACCCGCGCGTGGCGGGCGACGT CGGCAAAGCGGGCGTCGCTATCGACACCG TGGAAGATATGAAAGTCCTGTTCGACCAG ATCCCGCTGGATAAAATGTCGGTTTCGAT GACCATGAATGGCGCAGTGCTACCAGTAC TGGCGTTTTATATCGTCGCCGCAGAAGAG CAAGGTGTTACACCTGATAAACTGACCGG CACCATTCAAAACGATATTCTCAAAGAGTA CCTCTGCCGCAACACCTATATTTACCCACC AAAACCGTCAATGCGCATTATCGCCGACA TCATCGCCTGGTGTTCCGGCAACATGCCG CGATTTAATACCATCAGTATCAGCGGTTAC CACATGGGTGAAGCGGGTGCCAACTGCGT GCAGCAGGTAGCATTTACGCTCGCTGATG GGATTGAGTACATCAAAGCAGCAATCTCT GCCGGACTGAAAATTGATGACTTCGCTCC TCGCCTGTCGTTCTTCTTCGGCATCGGCAT GGATCTGTTTATGAACGTCGCCATGTTGC GTGCGGCACGTTATTTATGGAGCGAAGCG GTCAGTGGATTTGGCGCACAGGACCCGAA ATCACTGGCGCTGCGTACCCACTGCCAGA CCTCAGGCTGGAGCCTGACTGAACAGGAT CCGTATAACAACGTTATCCGCACCACCATT GAAGCGCTGGCTGCGACGCTGGGCGGTAC TCAGTCACTGCATACCAACGCCTTTGACGA AGCGCTTGGTTTGCCTACCGATTTCTCAGC ACGCATTGCCCGCAACACCCAGATCATCA TCCAGGAAGAATCAGAACTCTGCCGCACC GTCGATCCACTGGCCGGATCCTATTACATT GAGTCGCTGACCGATCAAATCGTCAAACA AGCCAGAGCTATTATCCAACAGATCGACG AAGCCGGTGGCATGGCGAAAGCGATCGAA GCAGGTCTGCCAAAACGAATGATCGAAGA GGCCTCAGCGCGCGAACAGTCGCTGATCG ACCAGGGCAAGCGTGTCATCGTTGGTGTC AACAAGTACAAACTGGATCACGAAGACGA AACCGATGTACTTGAGATCGACAACGTGA TGGTGCGTAACGAGCAAATTGCTTCGCTG GAACGCATTCGCGCCACCCGTGATGATGC CGCCGTAACCGCCGCGTTGAACGCCCTGA CTCACGCCGCACAGCATAACGAAAACCTG CTGGCTGCCGCTGTTAATGCCGCTCGCGT TCGCGCCACCCTGGGTGAAATTTCCGATG CGCTGGAAGTCGCTTTCGACCGTTATCTG GTGCCAAGCCAGTGTGTTACCGGCGTGAT TGCGCAAAGCTATCATCAGTCTGAGAAAT CGGCCTCCGAGTTCGATGCCATTGTTGCG CAAACGGAGCAGTTCCTTGCCGACAATGG TCGTCGCCCGCGCATTCTGATCGCTAAGA TGGGCCAGGATGGACACGATCGCGGCGCG AAAGTGATCGCCAGCGCCTATTCCGATCT CGGTTTCGACGTAGATTTAAGCCCGATGTT CTCTACACCTGAAGAGATCGCCCGCCTGG CCGTAGAAAACGACGTTCACGTAGTGGGC GCATCCTCACTGGCTGCCGGTCATAAAAC GCTGATCCCGGAACTGGTCGAAGCGCTGA AAAAATGGGGACGCGAAGATATCTGCGTG GTCGCGGGTGGCGTCATTCCGCCGCAGGA TTACGCCTTCCTGCAAGAGCGCGGCGTGG CGGCGATTTATGGTCCAGGTACACCTATG CTCGACAGTGTGCGCGACGTACTGAATCT GATAAGCCAGCATCATGATTAATGAAGCC ACGCTGGCAGAAAGTATTCGCCGCTTACG TCAGGGTGAGCGTGCCACACTCGCCCAGG CCATGACGCTGGTGGAAAGCCGTCACCCG CGTCATCAGGCACTAAGTACGCAGCTGCT TGATGCCATTATGCCGTACTGCGGTAACA CCCTGCGACTGGGCGTTACCGGCACCCCC GGCGCGGGGAAAAGTACCTTTCTTGAGGC CTTTGGCATGTTGTTGATTCGAGAGGGATT AAAGGTCGCGGTTATTGCGGTCGATCCCA GCAGCCCGGTCACTGGCGGTAGCATTCTC GGGGATAAAACCCGCATGAATGACCTGGC GCGTGCCGAAGCGGCGTTTATTCGCCCGG TACCATCCTCCGGTCATCTGGGCGGTGCC AGTCAGCGAGCGCGGGAATTAATGCTGTT ATGCGAAGCAGCGGGTTATGACGTAGTGA TTGTCGAAACGGTTGGCGTCGGGCAGTCG GAAACAGAAGTCGCCCGCATGGTGGACTG TTTTATCTCGTTGCAAATTGCCGGTGGCGG CGATGATCTGCAGGGCATTAAAAAAGGGC TGATGGAAGTGGCTGATCTGATCGTTATC AACAAAGACGATGGCGATAACCATACCAA TGTCGCCATTGCCCGGCATATGTACGAGA GTGCCCTGCATATTCTGCGACGTAAATAC GACGAATGGCAGCCACGGGTTCTGACTTG TAGCGCACTGGAAAAACGTGGAATCGATG AGATCTGGCACGCCATCATCGACTTCAAA ACCGCGCTAACTGCCAGTGGTCGTTTACA ACAAGTGCGGCAACAACAATCGGTGGAAT GGCTGCGTAAGCAGACCGAAGAAGAAGTA CTGAATCACCTGTTCGCGAATGAAGATTTC GATCGCTATTACCGCCAGACGCTTTTAGC GGTCAAAAACAATACGCTCTCACCGCGCA CCGGCCTGCGGCAGCTCAGTGAATTTATC CAGACGCAATATTTTGATTAAAGGAATTTT TATGTCTTATCAGTATGTTAACGTTGTCAC TATCAACAAAGTGGCGGTCATTGAGTTTAA CTATGGCCGAAAACTTAATGCCTTAAGTAA AGTCTTTATTGATGATCTTATGCAGGCGTT AAGCGATCTCAACCGGCCGGAAATTCGCT GTATCATTTTGCGCGCACCGAGTGGATCC AAAGTCTTCTCCGCAGGTCACGATATTCAC GAACTGCCGTCTGGCGGTCGCGATCCGCT CTCCTATGATGATCCATTGCGTCAAATCAC CCGCATGATCCAAAAATTCCCGAAACCGA TCATTTCGATGGTGGAAGGTAGTGTTTGG GGTGGCGCATTTGAAATGATCATGAGTTC CGATCTGATCATCGCCGCCAGTACCTCAA CCTTCTCAATGACGCCTGTAAACCTCGGC GTCCCGTATAACCTGGTCGGCATTCACAA CCTGACCCGCGACGCGGGCTTCCACATTG TCAAAGAGCTGATTTTTACCGCTTCGCCAA TCACCGCCCAGCGCGCGCTGGCTGTCGGC ATCCTCAACCATGTTGTGGAAGTGGAAGA ACTGGAAGATTTCACCTTACAAATGGCGC ACCACATCTCTGAGAAAGCGCCGTTAGCC ATTGCCGTTATCAAAGAAGAGCTGCGTGT ACTGGGCGAAGCACACACCATGAACTCCG ATGAATTTGAACGTATTCAGGGGATGCGC CGCGCGGTGTATGACAGCGAAGATTACCA GGAAGGGATGAACGCTTTCCTCGAAAAAC GTAAACCTAATTTCGTTGGTCATTAATCCC TGCGAACGAAGGAGTAAAAATGGAAACTCA GTGGACAAGGATGACCGCCAATGAAGCGG CAGAAATTATCCAGCATAACGACATGGTG GCATTTAGCGGCTTTACCCCGGCGGGTTC GCCGAAAGCCCTACCCACCGCGATTGCCC GCAGAGCTAACGAACAGCATGAGGCCAAA AAGCCGTATCAAATTCGCCTTCTGACGGG TGCGTCAATCAGCGCCGCCGCTGACGATG TACTTTCTGACGCCGATGCTGTTTCCTGGC GTGCGCCATATCAAACATCGTCCGGTTTAC GTAAAAAGATCAATCAGGGCGCGGTGAGT TTCGTTGACCTGCATTTGAGCGAAGTGGC GCAAATGGTCAATTACGGTTTCTTCGGCG ACATTGATGTTGCCGTCATTGAAGCATCG GCACTGGCACCGGATGGTCGAGTCTGGTT AACCAGCGGGATCGGTAATGCGCCGACCT GGCTGCTGCGGGCGAAGAAAGTGATCATT GAACTCAATCACTATCACGATCCGCGCGTT GCAGAACTGGCGGATATTGTGATTCCTGG CGCGCCACCGCGGCGCAATAGCGTGTCGA TCTTCCATGCAATGGATCGCGTCGGTACC CGCTATGTGCAAATCGATCCGAAAAAGAT TGTCGCCGTCGTGGAAACCAACTTGCCCG ACGCCGGTAATATGCTGGATAAGCAAAAT CCCATGTGCCAGCAGATTGCCGATAACGT GGTCACGTTCTTATTGCAGGAAATGGCGC ATGGGCGTATTCCGCCGGAATTTCTGCCG CTGCAAAGTGGCGTGGGCAATATCAATAA TGCGGTAATGGCGCGTCTGGGGGAAAACC CGGTAATTCCTCCGTTTATGATGTATTCGG AAGTGCTACAGGAATCGGTGGTGCATTTA CTGGAAACCGGCAAAATCAGCGGGGCCAG CGCCTCCAGCCTGACAATCTCGGCCGATT CCCTGCGCAAGATTTACGACAATATGGATT ACTTTGCCAGCCGCATTGTGTTGCGTCCG CAGGAGATTTCCAATAACCCGGAAATCAT CCGTCGTCTGGGCGTCATCGCTCTGAACG TCGGCCTGGAGTTTGATATTTACGGGCAT GCCAACTCAACACACGTAGCCGGGGTCGA TCTGATGAACGGCATCGGCGGCAGCGGTG ATTTTGAACGCAACGCGTATCTGTCGATCT TTATGGCCCCGTCGATTGCTAAAGAAGGC AAGATCTCAACCGTCGTGCCAATGTGCAG CCATGTTGATCACAGCGAACACAGCGTCA AAGTGATCATCACCGAACAAGGGATCGCC GATCTGCGCGGTCTTTCCCCGCTTCAACG CGCCCGCACTATCATTGATAATTGTGCACA TCCTATGTATCGGGATTATCTGCATCGCTA TCTGGAAAATGCGCCTGGCGGACATATTC ACCACGATCTTAGCCACGTCTTCGACTTAC ACCGTAATTTAATTGCAACCGGCTCGATGC TGGGTTAA

Next, this strain was tested for propionate production.

Briefly, 3 ml LB (containing selective antibiotics (cam) where necessary was inoculated from frozen glycerol stocks with either wild type E. coli K12 or the genetically engineered bacteria comprising the chromosomal sleeping beauty mutase operon under the control of a FNR promoter. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X10⁸ CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 1, 2, and 24 hours, 120 ul of cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for propionate concentrations, as described in

Results are depicted in FIG. 22B and show that the genetically engineered strain produces ˜2.5 mM after 24 h, while very little or no propionate production was detected from the E. coli K12 wild type strain. Propionate was measured as described in Example 26.

Example 24 Evaluation of the Sleeping Beauty Mutase Pathway for the Production of Propionate in E coli Nissle

Next, the SBM pathway is evaluated for propionate production in E. coli Nissle. Nissle does not have the full 4-gene sleeping beauty mutase operon; it only has the first gene and a partial gene of the second, and genes 3 and 4 are missing. Therefore, recombineering is used to introduce this pathway into Nissle. The frt-cam-frt-PfnrS-sbm, ygfD, ygfG, ygfH construct is inserted at the location of the endogenous, truncated Nissle SBM. Next, the construct is transformed into E coli Nissle and tested for propionate production essentially as described above.

Example 25 Evaluation of the Acrylate Pathway from Clostridium propionicum for Propionate Production

The acrylate pathway from Clostridium propionicum is evaluated for adaptation to propionate production in E. coli . A construct (Ptet-pct-1cdABC-acrABC), codon optimized for E. coli , was synthesized by Genewiz and placed in a high copy plasmid (Logic051). Additionally, another construct is generated for side by side testing, in which the acrABC genes (which may be the rate limiting step of the pathway) are replaced with the acuI gene from Rhodobacter sphaeroides (Ptet- acuI-pct-lcdABC). Subsequently these constructs are transformed into BW25113 and are assessed for their ability to produce propionate, as compared to the type BW5113 strain as described above in Example 23. Propionate was measured as described in Example 26.

Table 48 of Exemplary Propionate Cassette Sequences Description and SEQ ID NO Sequence Ptet-pct- ttaagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataaga lcdABC- aggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactat acrABC; Ptet: cagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaa lower case; gtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataa tertR/tetA aaaggctaattgattttcgagagtttcatactgatttctgtaggccgtgtacctaaatgtacttttgc promoter tccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgcca within Ptet: gctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgt lower case cgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg bold, with tet cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatca operator: ctttacttttatctaatctagacatcattaattcctaatttttgttgac actctatcattgatagagt t lower case attttaccac tccctatcagtgatagag aaaagtgaactctagaaataattttgtttaacttta bold a gaaggagatatacat ATGCGCAAAGTGCCGATTATCACGGCTG underlined; ACGAGGCCGCAAAACTGATCAAGGACGGCGACACCGTG ribosome ACAACTAGCGGCTTTGTGGGTAACGCGATCCCTGAGGCC binding site CTTGACCGTGCAGTCGAAAAGCGTTTCCTGGAAACGGGC and leader: GAACCGAAGAACATTACTTATGTATATTGCGGCAGTCAG lowe case GGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGCAT italic; GAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCG ribosome ACCGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAA binding sites: AATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGTGTCA lower case TCTCTTTCGCGATATTGCGAGCCATAAACCGGGTGTGTTC underlined; ACGAAAGTAGGAATCGGCACCTTCATTGATCCACGTAAC coding GGTGGTGGGAAGGTCAACGATATTACCAAGGAAGATAT regions: upper CGTAGAACTGGTGGAAATTAAAGGGCAGGAATACCTGTT case; (SEQ ID TTATCCGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGC NO: 239) ACCTATGCGGACGAGAGTGGTAACATCACCTTTGAAAAA GAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCG GTGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAG CGTGTCGTCAAAGCAGGCACGCTGGATCCGCGCCATGTG AAAGTTCCGGGTATCTATGTAGATTACGTAGTCGTCGCG GATCCGGAGGACCATCAACAGTCCCTTGACTGCGAATAT GATCCTGCCCTTAGTGGAGAGCACCGTCGTCCGGAGGTG GTGGGTGAACCACTGCCTTTATCCGCGAAGAAAGTCATC GGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACGTTGCA GTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCC GTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTC ACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGC GGTGTTCGCTTTGGGGCGAGCTACAATGCTGACGCCTTG ATCGACCAGGGCTACCAATTTGATTATTACGACGGTGGG GGTCTGGATCTTTGTTACCTGGGTTTAGCTGAATGCGACG AAAAGGGTAATATCAATGTTAGCCGCTTCGGTCCTCGTA TCGCTGGGTGCGGCGGATTCATTAACATTACCCAAAACA CGCCGAAAGTCTTCTTTTGTGGGACCTTTACAGCCGGGG GGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCG TTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTG GAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAAC AAGCAACAAGTTACCTACATCACGGAGCGTTGCGTCTTC CTCCTCAAAGAAGACGGTTTACACCTTTCGGAAATCGCG CCAGGCATCGATCTGCAGACCCAGATTTTGGATGTTATG GACTTTGCCCCGATCATTGATCGTGACGCAAACGGGCAG ATTAAACTGATGGACGCGGCGTTATTCGCAGAAGGGCTG ATGGGCTTGAAAGAAATGAAGTCTTGAtaagaaggagatatacatA TGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAG CATACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAG CGAAAGCCCGCGGTGAGCTGGTCTGCTGGTCGGCGTCAG TCGCGCCGCCGGAATTTTGCGTAACAATGGGCATTGCCA TGATCTACCCGGAGACTCATGCAGCGGGCATCGGTGCCC GCAAAGGTGCGATGGACATGCTGGAAGTTGCGGACCGC AAAGGCTACAACGTGGATTGTTGTTCCTACGGCCGTGTA AATATGGGTTACATGGAATGTTTAAAAGAAGCCGCCATC ACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCT GCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATA ATATCTGTAACACGCTGCTGAAATGGTACGAAAACTTAG CAGCAGAACTCGATATTCCTTGCATCGTGATCGACGTAC CGTTTAATCATACCATGCCGATTCCGGAATATGCCAAGG CCTACATCGCGGACCAGTTCCGCAATGCAATTTCTCAGC TGGAAGTTATTTGTGGCCGTCCGTTCGATTGGAAGAAAT TTAAGGAGGTCAAAGATCAGACCCAGCGTAGCGTATACC ACTGGAACCGCATTGCCGAGATGGCGAAATACAAGCCTA GCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTT AATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTAC CTTTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAA GGCGGGTATCTACGCCTTTAAAGGTGCGGAAAAAACGCG CTTTCAATGGGAAGGTATCGCGGTGTGGCCACATTTAGG TCACACGTTTAAATCTATGAAGAATCTGAATTCGATTAT GACCGGTACGGCATACCCCGCCCTTTGGGACCTGCACTA TGACGCTAACGACGAATCTATGCACTCTATGGCTGAAGC GTACACCCGTATTTATATTAATACTTGTCTGCAGAACAA AGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGT GGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACT GATGAGTTTCCTGAACGTGGAAACGGCTGAAATTATTAA AGAGAAGAACGGTCTTCCTTACGTCTCCATTGATGGCGA TCAGACCGATCCTCGCGTTTTTTCTCCGGCCCAGTTTGAT ACCCGTGTTCAGGCCCTGGTTGAGATGATGGAGGCCAAT ATGGCGGCAGCGGAATAAtaagaaggagatatacatATGTCACGC GTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGCCGCG AATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAAC AGGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCC CGAAGAAATGGTACACGCCGCTGGCTATTTGCCGATGGG AATCTGGGGCGCCCAGGGCAAAACGATTAGTAAAGCGC GCACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCA GGTTATGGAATTACAGTGCGAGGGCGCGTATGATGACCT GTCCGCAGTTATTTTTAGCGTACCGTGCGACACTCTCAAA TGTCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATT GTATTTACGCATCCGCAGAACCGCGGATTAGAAGCGGCG AACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCA CAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCC GCCCTGGAAAATTCGATTGCAATTTATAACGAGAATCGT GCCGTGATGCGTGAGTTCGTGAAAGTGGCAGCGGACTAT CCTCAAGTCATTGACGCAGTGAGCCGCCACGCGGTTTTT AAAGCGCGCCAGTTTATGCTTAAGGAAAAACATACCGCA CTTGTGAAAGAACTGATCGCTGAGATTAAAGCAACGCCA GTCCAGCCGTGGGACGGAAAAAAGGTTGTAGTGACGGG CATTCTGTTGGAACCGAATGAGTTATTAGATATCTTTAAT GAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAG GAAAGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAA GGCGGACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAA ATGTATGGCTGCTCGCTGGCAACCGACACCAAGAAGGGT CGCGGCCGTATGTTAATTAACAAAACGATTCAGACCGGT GCGGACGCTATCGTAGTTGCAATGATGAAGTTTTGCGAC CCAGAAGAATGGGATTATCCGGTAATGTACCGTGAATTT GAAGAAAAAGGGGTCAAATCACTTATGATTGAGGTGGA TCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCT GCAGTCATTCGTCGAAATGCTTTAAtaagaaggagatatacatATG TATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAG CGGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCG AGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAAC GCGCACTGGACAAAGCCTTTGAAGTCTCTGGCTTAAAAA AGGAAGACATCAGCTACACAGTAGCTACGGGCTATGGG CGCTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAA ATTAGCTGTCATGCCAAAGGCATTTATTTCTTAGTACCAA CTGCGCGCACTATTATTGACATTGGCGGCCAAGATGCGA AAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGCAA TTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGT TTCCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTG GATGAAATGGCTGAACTGGATGAACAGGCGACTGACAC CGCTCCCATTTCAAGCACCTGCACGGTTTTCGCCGAAAG CGAAGTAATTAGCCAATTGAGCAATGGTGTCTCACGCAA CAACATCATTAAAGGTGTCCATCTGAGCGTTGCGTCACG TGCGTGTGGTCTGGCGTATCGCGGCGGTTTGGAGAAAGA TGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGT GGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTAT CGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGC AGCGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTAAtaa gaaggagatatacatATGGCCTTCAATAGCGCAGATATTAATTCT TTCCGCGATATTTGGGTGTTTTGTGAACAGCGTGAGGGC AAACTGATTAACACCGATTTCGAATTAATTAGCGAAGGT CGTAAACTGGCTGACGAACGCGGAAGCAAACTGGTTGG AATTTTGCTGGGGCACGAAGTTGAAGAAATCGCAAAAG AATTAGGCGGCTATGGTGCGGACAAGGTAATTGTGTGCG ATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGC CAAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGA GGTAATTTTGATCGGTGCCACCAACATTGGCCGTGATCT CGGACCGCGTTGTGCTGCACGCTTGCACACGGGGCTGAC GGCTGATTGCACGCACCTGGATATTGATATGAATAAATA TGTGGACTTTCTTAGCACCAGTAGCACCTTGGATATCTCG TCGATGACTTTCCCTATGGAAGATACAAACCTTAAAATG ACGCGCCCTGCATTTGGCGGACATCTGATGGCAACGATC ATTTGTCCACGCTTCCGTCCCTGTATGAGCACAGTGCGCC CCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATG GCGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTG TCGGATGAAGACCTTAAAACTAAAGTAATTAATATCGTG AAGGAAACGAAAAAGATTGTGGATCTGATCGGCGCAGA AATTATTGTGTCAGTTGGTCGTGGTATCTCGAAAGATGTC CAAGGTGGAATTGCACTGGCTGAAAAACTTGCGGACGCA TTTGGTAACGGTGTCGTGGGCGGCTCGCGCGCAGTGATT GATTCCGGCTGGTTACCTGCGGATCATCAGGTTGGACAA ACCGGTAAGACCGTGCACCCGAAAGTCTACGTGGCGCTG GGTATTAGTGGGGCTATCCAGCATAAGGCTGGGATGCAA GACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACG GCGCCTATCTTCGACTGCGCCGATTATGGCATCACCGGT GATTTATTTAAAATCGTACCGATGATGATCGACGCGATC AAAGAGGGTAAAAACGCATGAtaagaaggagatatacatATGCGC ATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGGC AAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCC TCAATGGCAGCGATTATTAACCCGGACGATATGTCCGCG ATCGAACAGGCATTAAAACTGAAAGATGAAACCGGATG CCAGGTTACGGCGCTTACGATGGGTCCTCCTCCTGCCGA GGGCATGTTGCGCGAAATTATTGCAATGGGGGCCGACGA TGGTGTGCTGATTTCGGCCCGTGAATTTGGGGGGTCCGA TACCTTCGCAACCAGTCAAATTATTAGCGCGGCAATCCA TAAATTAGGCTTAAGCAATGAAGACATGATCTTTTGCGG TCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCC TCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTA TGGCGCAGGAATCAAAAAATCTGGTGATTTAGTGCTGGT GAAGCGTATGTTGGAGGATGGTTATATGATGATCGAAGT CGAAACTCCATGTCTGATTACCTGCATTCAGGATAAAGC GGTAAAACCACGTTACATGACTCTCAACGGTATTATGGA ATGCTACTCCAAGCCGCTCCTCGTTCTCGATTACGAAGC ACTGAAAGATGAACCGCTGATCGAACTTGATACCATTGG GCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTTTAC GCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCAC CGATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGA TGCAGAAACATGTCATCTAAtaagaaggagatatacatATGTTCTT ACTGAAGATTAAAAAAGAACGTATGAAACGCATGGACT TTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGG CGCGTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCG AAGAGATTGATCGTGAGCACGTTTTTCCTGCAGAAAACT TTAAGAAGATGGCGGAAATTGGCTTAACCGGCATTGGTA TCCCGAAAGAATTTGGTGGCTCCGGTGGAGGCACCCTGG AGAAGGTCATTGCCGTGTCAGAATTCGGCAAAAAGTGTA TGGCCTCAGCTTCCATTTTAAGCATTCATCTTATCGCGCC GCAGGCAATCTACAAATATGGGACCAAAGAACAGAAAG AGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAACTGG GCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATG CCGGCGCGGTAAAAACGACCGCGATTCTGGACAGCCAG ACAAACGAGTACGTGCTGAATGGCACCAAATGCTTTATC AGCGGGGGCGGGCGCGCGGGTGTTCTTGTAATTTTTGCG CTTACTGAACCGAAAAAAGGTCTGAAAGGGATGAGCGC GATTATCGTGGAGAAAGGGACCCCGGGCTTCAGCATCGG CAAGGTGGAGAGCAAGATGGGGATCGCAGGTTCGGAAA CCGCGGAACTTATCTTCGAAGATTGTCGCGTTCCGGCTG CCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAAATTG CTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCG CTCAAGCAATCGGAATTGCCGAGGGGGCGATCGACCTGA GTGTGAAGTACGTTCACGAGCGCATTCAATTTGGTAAAC CGATCGCGAATCTGCAGGGAATTCAATGGTATATCGCGG ATATGGCGACCAAAACCGCCGCGGCACGCGCACTTGTTG AGTTTGCAGCGTATCTTGAAGACGCGGGTAAACCGTTCA CAAAGGAATCTGCTATGTGCAAGCTGAACGCCTCCGAAA ACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATTCACG GGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGTA TGTATCGCGATGCTAAGATTACGGAAATTTACGAGGGGA CATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAA TGAAACGCTAA pct-lcdABC- ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCA acrABC AAACTGATCAAGGACGGCGACACCGTGACAACTAGCGG (ribosome CTTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGC binding sites: AGTCGAAAAGCGTTTCCTGGAAACGGGCGAACCGAAGA lower case ACATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCG underlined; ACGGTCGTGGCGCAGAACATTTCGCGCATGAAGGCCTGC coding TGAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGG regions: upper CGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCC case) (SEQ ID TACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCG NO: 240) ATATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAG GAATCGGCACCTTCATTGATCCACGTAACGGTGGTGGGA AGGTCAACGATATTACCAAGGAAGATATCGTAGAACTGG TGGAAATTAAAGGGCAGGAATACCTGTTTTATCCGGCGT TCCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGG ACGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCG CCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAAGAAC TCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTC AAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCG GGTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAG GACCATCAACAGTCCCTTGACTGCGAATATGATCCTGCC CTTAGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAA CCACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGT GGCGCGATTGAGCTCGAGAAAGACGTTGCAGTGAACCTT GGGGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGAT GAAGAAGGCATTGTGGATTTTATGACTCTCACAGCGGAG TCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGC TTTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAG GGCTACCAATTTGATTATTACGACGGTGGGGGTCTGGAT CTTTGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGT AATATCAATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGT GCGGCGGATTCATTAACATTACCCAAAACACGCCGAAAG TCTTCTTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAG TGAAAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAG GGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAAATC ACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAA GTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAG AAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCG ATCTGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCC GATCATTGATCGTGACGCAAACGGGCAGATTAAACTGAT GGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAA AGAAATGAAGTCTTGAtaagaaggagatatacatATGAGCTTAACC CAAGGCATGAAAGCTAAACAACTGTTAGCATACTTTCAG GGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCG CGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCC GGAATTTTGCGTAACAATGGGCATTGCCATGATCTACCC GGAGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTG CGATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTAC AACGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGT TACATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTC AAGCCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTC CGCTTCCCGATTTGGTGATTACGTGTAATAATATCTGTAA CACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACT CGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCAT ACCATGCCGATTCCGGAATATGCCAAGGCCTACATCGCG GACCAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTT GTGGCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCA AAGATCAGACCCAGCGTAGCGTATACCACTGGAACCGCA TTGCCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACG GCTTCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTG CCGCAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTT CGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATCT ACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGG AAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTA AATCTATGAAGAATCTGAATTCGATTATGACCGGTACGG CATACCCCGCCCTTTGGGACCTGCACTATGACGCTAACG ACGAATCTATGCACTCTATGGCTGAAGCGTACACCCGTA TTTATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCC TGCTTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCG TATATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCT GAACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACG GTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCC TCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAG GCCCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCG GAATAAtaagaaggagatatacatATGTCACGCGTGGAGGCAATCC TGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAA GCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGC GGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGT ACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCC TGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATT ACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTAT TTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAG AAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCAT CCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTG GTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCA GTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAAT TCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATT GACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAG TTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAA CTGATCGCTGAGATTAAAGCAACGCCAGTCCAGCCGTGG GACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAA CCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATC GCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCAG ATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTC TACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGC TCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATG TTAATTAACAAAACGATTCAGACCGGTGCGGACGCTATC GTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGG GATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGG GTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCG TCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCG AAATGCTTTAAtaagaaggagatatacatATGTATACCTTGGGGAT TGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAA AGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGT CGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAA AGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAG CTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAG CGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGC CAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTATT ATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTG GACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGAT AAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATG GCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAA CTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGC ACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAA TTGAGCAATGGTGTCTCACGCAACAACATCATTAAAGGT GTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCG TATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGT GGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGC GGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCC TCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTA TGAGGCCGCCCAGAAGAAGTAAtaagaaggagatatacatATGGC CTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTGG GTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACC GATTTCGAATTAATTAGCGAAGGTCGTAAACTGGCTGAC GAACGCGGAAGCAAACTGGTTGGAATTTTGCTGGGGCAC GAAGTTGAAGAAATCGCAAAAGAATTAGGCGGCTATGG TGCGGACAAGGTAATTGTGTGCGATCATCCGGAACTTAA ATTTTACACTACGGATGCTTATGCCAAAGTTTTATGTGAC GTCGTGATGGAAGAGAAACCGGAGGTAATTTTGATCGGT GCCACCAACATTGGCCGTGATCTCGGACCGCGTTGTGCT GCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCAC CTGGATATTGATATGAATAAATATGTGGACTTTCTTAGC ACCAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTA TGGAAGATACAAACCTTAAAATGACGCGCCCTGCATTTG GCGGACATCTGATGGCAACGATCATTTGTCCACGCTTCC GTCCCTGTATGAGCACAGTGCGCCCCGGAGTGATGAAGA AAGCGGAGTTCTCGCAGGAGATGGCGCAAGCATGTCAA GTAGTGACCCGTCACGTAAATTTGTCGGATGAAGACCTT AAAACTAAAGTAATTAATATCGTGAAGGAAACGAAAAA GATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGT TGGTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGC ACTGGCTGAAAAACTTGCGGACGCATTTGGTAACGGTGT CGTGGGCGGCTCGCGCGCAGTGATTGATTCCGGCTGGTT ACCTGCGGATCATCAGGTTGGACAAACCGGTAAGACCGT GCACCCGAAAGTCTACGTGGCGCTGGGTATTAGTGGGGC TATCCAGCATAAGGCTGGGATGCAAGACTCTGAACTGAT CATTGCCGTCAACAAAGACGAAACGGCGCCTATCTTCGA CTGCGCCGATTATGGCATCACCGGTGATTTATTTAAAATC GTACCGATGATGATCGACGCGATCAAAGAGGGTAAAAA CGCATGAtaagaaggagatatacatATGCGCATCTATGTGTGTGTG AAACAAGTCCCAGATACGAGCGGCAAGGTGGCCGTTAA CCCTGATGGGACCCTTAACCGTGCCTCAATGGCAGCGAT TATTAACCCGGACGATATGTCCGCGATCGAACAGGCATT AAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGC TTACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCG AAATTATTGCAATGGGGGCCGACGATGGTGTGCTGATTT CGGCCCGTGAATTTGGGGGGTCCGATACCTTCGCAACCA GTCAAATTATTAGCGCGGCAATCCATAAATTAGGCTTAA GCAATGAAGACATGATCTTTTGCGGTCGTCAGGCCATTG ACGGTGATACGGCCCAAGTCGGCCCTCAAATTGCCGAAA AACTGAGCATCCCACAGGTAACCTATGGCGCAGGAATCA AAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATGTTGG AGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTC TGATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTT ACATGACTCTCAACGGTATTATGGAATGCTACTCCAAGC CGCTCCTCGTTCTCGATTACGAAGCACTGAAAGATGAAC CGCTGATCGAACTTGATACCATTGGGCTTAAAGGCTCCC CGACGAATATCTTTAAATCGTTTACGCCGCCTCAGAAAG GCGTTGGTGTCATGCTCCAAGGCACCGATAAGGAAAAAG TCGAGGATCTGGTGGATAAGCTGATGCAGAAACATGTCA TCTAAtaagaaggagatatacatATGTTCTTACTGAAGATTAAAAA AGAACGTATGAAACGCATGGACTTTAGTTTAACGCGTGA ACAGGAGATGTTAAAAAAACTGGCGCGTCAGTTTGCTGA GATCGAGCTGGAACCGGTGGCCGAAGAGATTGATCGTG AGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATGGCGG AAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTG GTGGCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCG TGTCAGAATTCGGCAAAAAGTGTATGGCCTCAGCTTCCA TTTTAAGCATTCATCTTATCGCGCCGCAGGCAATCTACAA ATATGGGACCAAAGAACAGAAAGAGACGTACCTGCCGC GTCTTACCAAAGGTGGTGAACTGGGCGCCTTTGCGCTGA CAGAACCAAACGCCGGAAGCGATGCCGGCGCGGTAAAA ACGACCGCGATTCTGGACAGCCAGACAAACGAGTACGT GCTGAATGGCACCAAATGCTTTATCAGCGGGGGCGGGCG CGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAA AAAGGTCTGAAAGGGATGAGCGCGATTATCGTGGAGAA AGGGACCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCA AGATGGGGATCGCAGGTTCGGAAACCGCGGAACTTATCT TCGAAGATTGTCGCGTTCCGGCTGCCAACCTTTTAGGTA AAGAAGGCAAAGGCTTTAAAATTGCTATGGAAGCCCTGG ATGGCGCCCGTATTGGCGTGGGCGCTCAAGCAATCGGAA TTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGTTC ACGAGCGCATTCAATTTGGTAAACCGATCGCGAATCTGC AGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAA CCGCCGCGGCACGCGCACTTGTTGAGTTTGCAGCGTATC TTGAAGACGCGGGTAAACCGTTCACAAAGGAATCTGCTA TGTGCAAGCTGAACGCCTCCGAAAACGCGCGTTTTGTGA CAAATTTAGCTCTGCAGATTCACGGGGGTTACGGTTATA TGAAAGATTATCCGTTAGAGCGTATGTATCGCGATGCTA AGATTACGGAAATTTACGAGGGGACATCAGAAATCCATA AGGTGGTGATTGCGCGTGAAGTAATGAAACGCTAA Ptet-acuI-pct- caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggg lcdABC gatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaac (Ptet: lower gacggccagtgaattgacgcgtattgggatgtaaaacgacggccagtgaattcgttaagaccc case; tetA/R actttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataagaaggctggc promoter tctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagta within Ptet: ggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatg lower case ccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgaggcataaaaaggcta bold, with tet attgattttcgagagtttcatactgatttctgtaggccgtgtacctaaatgtacttttgctccatcgc operator gatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttcccc underlined; ttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaa RBS and gcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttac leader region gggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttat lower case ctaatctagacatcattaattcctaatttttgttgac actctatcattgatagagt tattttaccac italic; tccctatcagtgatagaga aaagtgaactctagaaataattttgtttaactttaa gaaggaga ribosome tatacat ATGCGTGCGGTACTGATCGAGAAGTCCGATGATAC binding site: ACAGTCCGTCTCTGTCACCGAACTGGCTGAAGATCAACT lower case GCCGGAAGGCGACGTTTTGGTAGATGTTGCTTATTCAAC underlined ACTGAACTACAAAGACGCCCTGGCAATTACCGGTAAAGC italic; coding CCCCGTCGTTCGTCGTTTTCCGATGGTACCTGGAATCGAC region: upper TTTACGGGTACCGTGGCCCAGTCTTCCCACGCCGACTTCA case, rrnB T1 AGCCAGGTGATCGCGTAATCCTGAATGGTTGGGGTGTGG and T2 GGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCGC terminors: GTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTG lower case GACTTACGCCAAGCGGCCATGATCGGTACAGCAGGATAC bold underline ACGGCGATGTTGTGCGTTCTGGCGCTTGAACGTCACGGA italics) (SEQ GTGGTGCCGGGTAATGGGGAAATCGTGGTGTCCGGTGCA ID NO: 241) GCAGGCGGCGTCGGCTCCGTTGCGACGACCCTTCTTGCC GCTAAGGGCTATGAGGTAGCGGCAGTGACTGGACGTGC GTCCGAAGCAGAATATCTGCGCGGTTTGGGGGCGGCGAG CGTAATTGATCGTAACGAATTAACGGGGAAGGTACGCCC GCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGC GGGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAA GTATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGG CATGGATCTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGT GGGATGACGCTGGCAGGGGTGGATAGCGTTATGTGCCCA AAGACAGATCGTTTAGCAGCGTGGGCCCGTTTGGCGTCA GATCTTGACCCTGCCAAGCTGGAGGAGATGACTACAGAG TTGCCGTTTAGTGAAGTAATCGAGACAGCACCCAAATTC TTGGACGGGACGGTTCGTGGCCGCATTGTTATCCCCGTA ACGCCCTAAgaactctagaaataattttgtttaactttaa gaaggagatatacat AT GCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAA AACTGATCAAGGACGGCGACACCGTGACAACTAGCGGC TTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGCA GTCGAAAAGCGTTTCCTGGAAACGGGCGAACCGAAGAA CATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCGA CGGTCGTGGCGCAGAACATTTCGCGCATGAAGGCCTGCT GAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGGC GTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCCT ACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGA TATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGG AATCGGCACCTTCATTGATCCACGTAACGGTGGTGGGAA GGTCAACGATATTACCAAGGAAGATATCGTAGAACTGGT GGAAATTAAAGGGCAGGAATACCTGTTTTATCCGGCGTT CCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGGA CGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCC TTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAAGAACTC GGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAA AGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGG TATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGA CCATCAACAGTCCCTTGACTGCGAATATGATCCTGCCCTT AGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACC ACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGG CGCGATTGAGCTCGAGAAAGACGTTGCAGTGAACCTTGG GGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGA AGAAGGCATTGTGGATTTTATGACTCTCACAGCGGAGTC CGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTT TGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGGG CTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTT TGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAAT ATCAATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGTGCG GCGGATTCATTAACATTACCCAAAACACGCCGAAAGTCT TCTTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAGTGA AAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGA AACAGAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCT TTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTTA CCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAG ACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATC TGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGAT CATTGATCGTGACGCAAACGGGCAGATTAAACTGATGGA CGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGA AATGAAGTCTTGAtaa gaaggagatatacat ATGAGCTTAACCCA AGGCATGAAAGCTAAACAACTGTTAGCATACTTTCAGGG TAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCG GTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGG AGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCG ATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAA CGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTAC ATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAG CCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGC TTCCCGATTTGGTGATTACGTGTAATAATATCTGTAACAC GCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGA TATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACC ATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGAC CAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTG GCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCAAAG ATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTG CCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACGGCT TCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCG CAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGC GGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTACG CCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAG GTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAAT CTATGAAGAATCTGAATTCGATTATGACCGGTACGGCAT ACCCCGCCCTTTGGGACCTGCACTATGACGCTAACGACG AATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTT ATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGC TTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCGTAT ATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGA ACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACGGT CTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTC GCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGC CCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGG AATAAtaa gaaggagatatacat ATGTCACGCGTGGAGGCAATCC TGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAA GCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGC GGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGT ACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCC TGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATT ACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTAT TTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAG AAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCAT CCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTG GTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCA GTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAAT TCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATT GACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAG TTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAA CTGATCGCTGAGATTAAAGCAACGCCAGTCCAGCCGTGG GACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAA CCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATC GCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCGG ATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTC TACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGC TCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATG TTAATTAACAAAACGATTCAGACCGGTGCGGACGCTATC GTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGG GATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGG GTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCG TCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCG AAATGCTTTAAtaa gaaggagatatacat ATGTATACCTTGGGGAT TGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAA AGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGT CGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAA AGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAG CTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAG CGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGC CAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTATT ATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTG GACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGAT AAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATG GCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAA CTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGC ACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAA TTGAGCAATGGTGTCTCACGCAACAACATCATTAAAGGT GTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCG TATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGT GGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGC GGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCC TCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTA TGAGGCCGCCCAGAAGAAGTAgatggtagtgtggggtctccccatgcga gagtagggaactgccaggcat

ccgccgggagcggattt gaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgcca ggcatcaaattaagc

acuI-pct- ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAG lcdABC (SEQ TCCGTCTCTGTCACCGAACTGGCTGAAGATCAACTGCCG ID NO: 242) GAAGGCGACGTTTTGGTAGATGTTGCTTATTCAACACTG AACTACAAAGACGCCCTGGCAATTACCGGTAAAGCCCCC GTCGTTCGTCGTTTTCCGATGGTACCTGGAATCGACTTTA CGGGTACCGTGGCCCAGTCTTCCCACGCCGACTTCAAGC CAGGTGATCGCGTAATCCTGAATGGTTGGGGTGTGGGGG AAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCGCGTGC GCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGACTT ACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGG CGATGTTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGG TGCCGGGTAATGGGGAAATCGTGGTGTCCGGTGCAGCAG GCGGCGTCGGCTCCGTTGCGACGACCCTTCTTGCCGCTA AGGGCTATGAGGTAGCGGCAGTGACTGGACGTGCGTCCG AAGCAGAATATCTGCGCGGTTTGGGGGCGGCGAGCGTA ATTGATCGTAACGAATTAACGGGGAAGGTACGCCCGCTG GGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGCGGG ATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGTA TCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCAT GGATCTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGTGGG ATGACGCTGGCAGGGGTGGATAGCGTTATGTGCCCAAAG ACAGATCGTTTAGCAGCGTGGGCCCGTTTGGCGTCAGAT CTTGACCCTGCCAAGCTGGAGGAGATGACTACAGAGTTG CCGTTTAGTGAAGTAATCGAGACAGCACCCAAATTCTTG GACGGGACGGTTCGTGGCCGCATTGTTATCCCCGTAACG CCCTAAgaactctagaaataattttgtttaactttaa gaaggagatatacat ATGCG CAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACT GATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGT GGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGA AAAGCGTTTCCTGGAAACGGGCGAACCGAAGAACATTA CTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGTC GTGGCGCAGAACATTTCGCGCATGAAGGCCTGCTGAAAC GTTATATCGCTGGCCATTGGGCGACCGTCCCGGCGTTAG GGAAAATGGCCATGGAGAATAAAATGGAGGCCTACAAT GTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATATTG CGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCG GCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCA ACGATATTACCAAGGAAGATATCGTAGAACTGGTGGAA ATTAAAGGGCAGGAATACCTGTTTTATCCGGCGTTCCCG ATCCATGTCGCGCTGATTCGTGGCACCTATGCGGACGAG AGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCCTTTG GAAGGGACTTCTGTCTGTCAAGCGGTGAAGAACTCGGGT GGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAAAGCA GGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTATC TATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCAT CAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGT GGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACCACT GCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGGCGC GATTGAGCTCGAGAAAGACGTTGCAGTGAACCTTGGGGT AGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGAAGA AGGCATTGTGGATTTTATGACTCTCACAGCGGAGTCCGG CGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGG GGCGAGCTACAATGCTGACGCCTTGATCGACCAGGGCTA CCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGT TACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATC AATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGC GGATTCATTAACATTACCCAAAACACGCCGAAAGTCTTC TTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAGTGAAA ATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGAAA CAGAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCTTT AATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTTAC CTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGA CGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCT GCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGAT CATTGATCGTGACGCAAACGGGCAGATTAAACTGATGGA CGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGA AATGAAGTCTTGAtaa gaaggagatatacat ATGAGCTTAACCCA AGGCATGAAAGCTAAACAACTGTTAGCATACTTTCAGGG TAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCG GTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGG AGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCG ATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAA CGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTAC ATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAG CCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGC TTCCCGATTTGGTGATTACGTGTAATAATATCTGTAACAC GCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGA TATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACC ATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGAC CAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTG GCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCAAAG ATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTG CCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACGGCT TCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCG CAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGC GGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTACG CCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAG GTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAAT CTATGAAGAATCTGAATTCGATTATGACCGGTACGGCAT ACCCCGCCCTTTGGGACCTGCACTATGACGCTAACGACG AATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTT ATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGC TTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCGTAT ATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGA ACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACGGT CTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTC GCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGC CCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGG AATAAtaa gaaggagatatacat ATGTCACGCGTGGAGGCAATCC TGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAA GCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGC GGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGT ACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCC TGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATT ACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTAT TTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAG AAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCAT CCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTG GTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCA GTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAAT TCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATT GACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAG TTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAA CTGATCGCTGAGATTAAAGCAACGCCAGTCCAGCCGTGG GACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAA CCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATC GCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCGG ATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTC TACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGC TCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATG TTAATTAACAAAACGATTCAGACCGGTGCGGACGCTATC GTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGG GATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGG GTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCG TCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCG AAATGCTTTAAtaa gaaggagatatacat ATGTATACCTTGGGGAT TGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAA AGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGT CGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAA AGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAG CTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAG CGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGC CAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTATT ATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTG GACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGAT AAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATG GCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAA CTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGC ACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAA TTGAGCAATGGTGTCTCACGCAACAACATCATTAAAGGT GTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCG TATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGT GGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGC GGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCC TCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTA TGAGGCCGCCCAGAAGAAGTA

Example 26 Quantification of Propionate by LC-MS/MS Sample Preparation

First, fresh 1000, 500, 250, 100, 20, 4 and 0.8 m/mL sodium propionate standards were prepared in water. Then, 25 μL of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 75 μL of 60% ACN (45 uL ACN+30 uL water per reaction) with 10 ug/mL of propionate-d5 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000 rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 5 μL of diluted samples were added to 95 μL of a buffer containing 10 mM MES pH4.5, 20 mM EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), and 20 mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour

LC-MS/MS Method

Propionate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 49 and Table 50. Tandem Mass Spectrometry details are found in Table 51.

TABLE 49 HPLC Details Column Thermo Aquasil C18 column, 5 μm (50 × 2.1 mm) Mobile 100% H2O, 0.1% Formic Phase A Acid Mobile 100% ACN, 0.1% Formic Phase B Acid Injection 10 uL volume

TABLE 50 HPLC Method Total Flow Time Rate (min) (μL/min) A % B % 0 0.5 100 0 1 0.5 100 0 2 0.5 10 90 4 0.5 10 90 4.01 0.5 100 0 4.25 0.5 100 0

TABLE 51 Tandem Mass Spectrometry Details Ion Source HESI-II Polarity Positive SRM Propionate transitions 156.2/57.1, Propionate-d5 161/62.1

Example 27 GLP-1 Production from Genetically Engineered Bacteria and Activity Measurements

To determine whether GLP-1 can be expressed by the genetically engineered bacteria, a construct expressing GLP-1 in conjunction with a modified flagellar type III secretion system shown in FIG. 61 was generated and integrated into the E coli Nissle chromosome. The construct comprises GLP-1 under control of the native FliC promoter and 5′UTR (untranslated region containing the N-terminal flagellar secretion signal) with an optimized ribosome binding site FIG. 23 and Table 52).

TABLE 52 GLP-1 construct sequences Description and SEQ ID NO Sequence GLP-1 under control of the native ttaaccacgacctttaaccagccaagcaataaactctttcgcagc FliC promoter and 5′UTR with an ctggccctccaaatagctagaaacatcagaagtgaaagttccct optimized ribosome binding site (in ccgcgtggcgttcgaactcgtccatattacctcctgactgtgtcta reverse orientation) cttcgttgattacgttttgggtttccacccgtcggctcaatcgccgt (SEQ ID NO: 243) ca GLP-1 (in reverse orientation) ttaaccacgacctttaaccagccaagcaataaactctttcgcagc (SEQ ID NO: 244) ctggccctccaaatagctagaaacatcagaagtgaaagttccct ccgcgtggcgttcgaactcgtccat FliC 5′ UTR (in reverse orientation) attacctcctgactgtgtctacttcgttgattacgttttgggtttcca (SEQ ID NO: 245) cccgtcggctcaatcgccgtca Optimized RBS (in reverse attacctcctgactgtgtctacttc orientation)  (SEQ ID NO: 246) Putative terminator gggcagaaaaaaccccgccgttggcggggaagcacgttgc (SEQ ID NO: 247) GLP-1 construct comprising Gggcagaaaaaaccccgccgttggcggggaagcacgttgc terminator (lower case italic) GLP-1 tggcaaattaccattcatgttgccggatgcggcgtaaacgcctta (lower case bold) under control of tccggcctacaaaaatgtgcaaattcaataaattgcaattcccctt the native FliC promoter and 5′UTR gtaggcctgataagcgcagcgcatcaggcaatttggcgttgcc (upper case bold, with optimized gtcagtctcagttaatcaggttacggcgattaaccacgaccttt RBS underlined) and a aaccagccaagcaataaactctttcgcagcctggccctcca chloramphenicol resistance gene aatagctagaaacatcagaagtgaaagttccctccgcgtgg under the control of the cat promoter cgttcgaactcgtccat ATTACCTCCTGACTGTG (upper case italic bold), frt homology TCTACTTCGTTGATTACGTTTTGGGTTT (upper case underlined) CCACCCGTCGGCTCAATCGCCGTCAAC (SEQ ID NO: 248) CCTGTTATCGTCTGTCGTAAAACAACC TTTAGAATTTTTTTCACAAACAGCCATT TTTTGTTAGTCGACGAAATACTCTTTTC TCTGCCCCTTATTCCCGCTATTAAAAAA AACAATTAAACGTAAACTTTGCGCAAT TCAGGCCGATAACCCCGGTATTCGTTT TACGTGTCGAAAGATAAACGAAGTTCC TATACTTTCTAGAGAATAGGAACTTCG GAATAGGAACTTCATTTCTCGTTCGCT GCCACCTAAGAATACTCTACGGTCACA TACAAATGGCGCGCCTTACGCCCCGCC CTGCCA

ACG TCTCATTTTCGCCAAAAGTTGGCCCAG GGCTTCCCGGTATCAACAGGGACACCA GGATTTATTTATTCTGCGAAGTGATCTT CCGTCACAGGTAGGCGCGCCGAAGTTC CTATACTTTCTAGAGAATAGGAACTTC GGAATAGGAACT

Cultures (the genetically engineered bacteria comprising the GLP-1 construct or streptomycin resistant control Nissle) are grown overnight in F-12K medium (Mediatech, Manassas, Va.) without glucose (containing selective antibiotics (chloramphenicol or streptomycin) and then diluted 1:200. The cells are grown with shaking at 250 rpm, and at indicated times (0, 3, 6, and 12 h), the supernatant aliquots are collected for GLP-1 quantification.

Additionally, bacteria are pelleted, washed, and harvested, resuspended in 25 mL sonication buffer (50 mM Tris-HCl, 30 mM NaCl, pH 8.0) with protease inhibitors, and lysed by sonication on ice. Insoluble debris is spun down twice for 20 min at 12,000 rpm at 4° C. to detect any intracellular recombinant protein.

To generate cell free medium, the supernatant is centrifuged, and filtered through a 0.2-micron filter to remove any remaining bacteria. The cell-free culture medium (CFM) is diluted to OD600=1 with F-12K, and 10 ng/ml leupeptin, 200 μM PMSF and 5 ng/mL aprotinin was added to the CFM to inhibit proteases prior storage at 4° C.

Western Blotting

The cell-free culture medium (CFM) was diluted to the same OD600 with F-12K, and 10 ng/ml leupeptin, PMSF and 5 ng/mL aprotinin was added to inhibit proteases. Clarified supernatant (14 ml) is precipitated with 10% trichloroacetic acid (TCA, VWR) for 30 min on ice, and the pellet was washed twice in ice-cold ethanol/ether (1:1). The supernatant pellet is dried under vacuum, dissolved in 50 pi sample buffer (2% SDS, 50 mM Tris, pH 6.8, 20%glycerol, 10% mercaptoethanol, bromophenol blue) and boiled for 5 min at 95° C. The cell pellet is resuspended (From 14 ml culture) in room temperature BugBuster Master Mix by gentle vortexing, using 500 μl BugBuster Master Mix with protease inhibitors (10 ng/ml Leupeptin, 200₁IMPMSF and 5 ng/mL aprotinin). The cell suspension is incubated on a shaking platform (VWR, Bristol, Conn.) at a slow setting for 10-20 min at room temperature. 125 μl 5× sample buffer is added to each sample before and boiling for 10 min at 95° C.

Protein concentration is determined by BCA protein assay, and isolated proteins are analyzed by Western blot. Proteins are transferred onto PVDF membranes are detected with an HRP-conjugated Glucagon Antibody (24HCLC), ABfinity™ Rabbit Oligoclonal, Thermo Fisher.

Co-culture with Caco-2 cells and ELISA for Insulin

To determine whether the GLP-1 expressed by the genetically engineered bacteria is functional, a co-culture experiment is conducted in which the bacterial supernatant containing GLP-1 is added to the growth medium of a mammalian intestinal cell line, Caco-2. Caco-2 cells are an intestinal cell line derived from a human colorectal carcinoma that spontaneously differentiates under standard culture conditions, and which lends itself to the in vitro study of human gut. The ability of the Caco-2 cells to produce insulin upon exposure to the bacterial cell free supernatant is measured.

Caco-2 epithelial cells (ATCC# CRL-2102, Manassas, Va.) are maintained in Dulbecco's Modified Eagle Media (DMEM, Cellgro, Herndon,Va.) plus 10% FBS (Cellgro) at 37° C. in a humidified incubator supplemented with 5% CO2. For co-culture experiments, Caco-2 cells are grown in F-12K supplemented with 10% FBS at 37° C. in a humidified incubator supplemented with 5% CO2. All co-culture experiments are performed in F-12K plus 10% FBS with Caco-2 cells in passages between 15 and 22.

Approximately 80% confluent monolayers of Caco-2 cells in 12-well plates are washed with fresh F-12K plus 10% FBS once and covered with 1 mL 50% CFM in F-12K with 10% FBS and incubated at 37° C. with 5% CO₂. 200 nM. As a control, the same volume of recombinant GLP-1 (200 nM) in F-12K with 10% FBS is added as a positive control in separate wells. Following a 16 h incubation, an additional 1 mL of 50% CFM in F-12K with 10% FBS or GLP-1 is added to the cells, supplemented with 0.4% Glucose or 0.4% Glycerol before incubation for an additional 2 h. The media is removed from the cells, supplemented with Leupeptin (10 ng/mL), 0.2 mM PMSF and aprotinin (10 ng/mL), centrifuged (12,000×rpm), and kept briefly at 4° C. prior to ELISA analysis for insulin expression (see “Immuno-blot and ELISA” section).

In order to estimate the amount of insulin secreted from Caco-2 cells activated by Glp-1, cell free supernatants are assayed using standard ELISA procedures using the Insulin ELISA Kit, Human (KAQ125, Thermo Fisher), according to manufacturer's instructions.

Example 28 In Vivo NASH Studies

For in vivo studies, a mouse model is used to study the effects of liver steatosis and hepatic inflammation (Jun Jin, et al., Brit. J. Nutrition, 114:145-1755 (2015)). To briefly summarize, female C57BL/6J mice are fasted and fed either a standard liquid diet of carbohydrates, fat, and protein; or a liquid Western style diet (WSD) fortified with fructose, fat, cholesterol, and a sodium butyrate supplement for six weeks. Butyrate is a short chain fatty acid naturally produced by intestinal bacteria effective in maintaining intestinal homoeostasis. Body weight and plasma samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and the liver and intestine can be removed and assayed. A decrease in liver damage after treatment with the engineered bacterial cells indicates that the engineered bacterial cells described herein are effective for treating nonalcoholic steatohepatitis (NASH).

Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with nonalcoholic steatohepatitis (NASH), for example, weight loss, further indicates the efficacy of the engineered bacterial cells described herein for treating nonalcoholic steatohepatitis (NASH).

Example 29 Construction of Plasmids Encoding Bile Salt Hydrolase Enzymes

The bile salt hydrolase genes from Lactobacillus plantarum (SEQ ID NO:1) is synthesized (Genewiz), fused to the Tet promoter, cloned into the high-copy plasmid pUC57-Kan by Gibson assembly, and transformed into E. coli DH5α as described herein to generate the plasmid pTet-BSH.

Example 30 Generation of Recombinant Bacteria Comprising a Bile Salt Hydrolase Enzyme

The pTet-BSH plasmid described above is transformed into E. coli Nissle, DH5α, or PIR1. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli (Nissle, DH5α or PIR1) is diluted 1:100 in 4 mL of LB and grown until it reaches an OD₆₀₀ of 0.4-0.6. 1mL of the culture is then centrifuged at 13,000 rpm for 1 min in a 1.5 mL microcentrifuge tube and the supernatant is removed. The cells are then washed three times in pre-chilled 10% glycerol and resuspended in 40 uL pre-chilled 10% glycerol. The electroporator is set to 1.8 kV. 1 uL of a pTet-BSH miniprep is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled 1mm cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 500 uL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C. for 1 hr. The cells are spread out on an LB plate containing 50 ug/mL Kanamycin for pTet-BSH.

Example 31 Functional Assay Demonstrating that the Recombinant Bacterial Cells Decrease Bile Salt Concentration

For in vitro studies, all incubations will be performed at 37° C. Cultures of E. coli Nissle containing pTet-BSH are grown overnight in LB and then diluted 1:100 in LB. The cells are grown with shaking (250 rpm) to early log phase with the appropriate antibiotics. Anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of bile salt hydrolase, and bacteria are grown for another 3 hours. Culture broths are then inoculated at 20% in flasks containing fresh LB culture media containing excess bile salts (either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile) and grown for 16 hours with shaking (250 rpm). A “medium blank” for each culture condition broth is also prepared whereby the “medium blank” is not inoculated with bacteria but treated under the same conditions as the inoculated broths. Following the 16 hour incubation period, broth cultures are pasteurized at 90° C. for 15 minutes, centrifuged at 5,000 rpm for 10 minutes, and supernatants filtered with a 0.45 micron filter.

Bile salt levels and activity in the supernatants is determined. Briefly, bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and Feighner, Applied Environ. Microbiol., 55:11-16 (1989) and Christiaens et al., Appl. Environ. Microbiol., 58:3792-3798 (1992). BSH activity can also be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al., PNAS, 105(36):13580-13585 (2008)). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al., Gut Microbes, 3(3):186-202 (2012)).

Example 32 In Vivo Studies Demonstrating that the Recombinant Bacterial Cells Decrease Bile Salt Concentration

For in vivo studies, a mouse model of weight gain and lipid metabolism (as described by Joyce et al., PNAS, 111(20):7421-7426 (2014)) is used. To briefly summarize, C57BL/6J mice and germ-free Swiss Webster mice can be fasted and fed either a normal low-fat diet or a high-fat diet for ten weeks. After ten weeks, the mice can be inoculated with recombinant bacteria comprising a bile salt hydrolase enzyme (as described herein) or control bacteria. Body weight, plasma samples, and fecal samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and internal organs (liver, spleen, intestines) and fat pads can be removed and assayed. Treatment efficacy is determined, for example, by measuring levels of bile salts and bile acids. A decrease in levels of bile salts after treatment with the recombinant bacterial cells indicates that the recombinant bacterial cells described herein are effective for treating disorders associated with bile salts.

Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with disorders associated with bile salts, for example, weight loss, further indicates the efficacy of the recombinant bacterial cells described herein for treating disorders associated with bile salts.

Example 33 Nitric oxide-Inducible Reporter Constructs

ATC and nitric oxide-inducible reporter constructs were synthesized (Genewiz, Cambridge, Mass.). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations (FIG. 77); promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide-inducible reporter construct is shown. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS are

TABLE 53 SEQ ID NO: 249 ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgt tgagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgcaccg ccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgggcccat acactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgtt cgggcggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccg cctttgaccagcgcggtaaccactttcatcaaatggcttttggaaatgccgtaggtcga ggcgatggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagatgaggacgc

aattttaaactctagaaataattttgtttaactttaagaaggagatatacata tggcta gcaaaggcgaagaattgttcacgggcgttgttcctattttggttgaattggatggcgat gttaatggccataaattcagcgttagcggcgaaggcgaaggcgatgctacgtatggcaa attgacgttgaaattcatttgtacgacgggcaaattgcctgttccttggcctacgttgg ttacgacgttcagctatggcgttcaatgtttcagccgttatcctgatcatatgaaacgt catgatttcttcaaaagcgctatgcctgaaggctatgttcaagaacgtacgattagctt caaagatgatggcaattataaaacgcgtgctgaagttaaattcgaaggcgatacgttgg ttaatcgtattgaattgaaaggcattgatttcaaagaagatggcaatattttgggccat aaattggaatataattataatagccataatgtttatattacggctgataaacaaaaaaa tggcattaaagctaatttcaaaattcgtcataatattgaagatggcagcgttcaattgg ctgatcattatcaacaaaatacgcctattggcgatggccctgttttgttgcctgataat cattatttgagcacgcaaagcgctttgagcaaagatcctaatgaaaaacgtgatcatat ggttttgttggaattcgttacggctgctggcattacgcatggcatggatgaattgtata aa taataa

These constructs, when induced by their cognate inducer, lead to high level expression of GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600=˜0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and 2-fold decreases in inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). It was observed that both the ATC and NO were able to induce the expression of GFP in their respective construct across a wide range of concentrations. Promoter activity is expressed as relative florescence units.

FIG. 77 shows NO-GFP constructs (the dot blot) E. coli Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1:100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria were resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using

Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS-treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation.

Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1:100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. FIG. 77D shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice.

Example 34 FNR Promoter Activity

In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 18 and Table 19. The nucleotide sequences of these constructs are shown in Tables 54-58 ((SEQ ID NO: 250-254). However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity, exemplary results are shown in FIG. 75.

Table 54 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_(fnr1) (SEQ ID NO: 250). The construct comprises a translational fusion of the Nissle nirB1 gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^(th) codon of the lacZ coding region. The P_(fnri) sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 55 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_(fnr2) ((SEQ ID NO: 251). The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^(th) codon of the lacZ coding region. The P_(fnr2) sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 56 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_(fnr3) ((SEQ ID NO: 252). The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The P_(fnr3) sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 57 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_(fnr4) ((SEQ ID NO: 253). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The P_(fnr4) sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 58 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS ((SEQ ID NO: 254). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrS1, fused to lacZ. The P_(fnrs) sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

TABLE 54 Pfnr1-lacZ construct Sequences Nucleotide sequences of Pfnr1-lacZ construct, low-copy (SEQ ID NO: 250) GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcacta tcgtcgtccggccttttcctctcttactctgctacgtacatctatttctataaatcc gttcaatttgtctgttttttgcacaaacatgaaatatcagacaattccgtgacttaa gaaaatttatacaaatcagcaatataccccttaaggagtatataaaggtgaatttga tttacatcaataagcggggttgctgaatcgttaaggtaggcggtaatag aaaagaaa tcgaggcaaaa ATGagcaaagtcagactcgcaattatGGATCCTCTGGCCGTCGTAT TACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCAC ATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCC AACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAG CGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCC CCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATC CCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGC TCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTG ATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCC AGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAA ACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGG ATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCA CGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGG TACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGG TTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTG AAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTG AAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAAC TGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCG AGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCG GCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGA TGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGC ATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGG TGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATG ATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATC GTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTA ATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGT ATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGC GCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGC TTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGG GTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTT TACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAA ACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCC AGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGG AAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAG TGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGG CACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAG GTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCT GGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACA TCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCG CGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGG GTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTG GCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGG ATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAAC GCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAG ATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGA AAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCA TCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGA CCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAG AAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGT CAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGC GCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCC GCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAG AAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCT GGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGT TGGTCTGGTGTCAAAAATAA

TABLE 55 Pfnr2-lacZ construct sequences Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 251) GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctc atgcatgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacag gagtatttatattgcgcccgttacgtgggcttcgactgtaaatc agaaaggagaaaaca cct ATGacgacctacgatcgGGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGA AAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGC GTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTG CGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACG ATGCGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCC GCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACA GGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCA ACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGC GCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGG CAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGT TGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGAT GATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGA ACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGC CTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGC CTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGT GGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTT TCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATT CGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGAC GATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGC ATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTG GATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCC GCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATC ACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGAC GCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGG CGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAG ACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGA GAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGG CTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCT GGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCT TACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGT CTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATT TCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCAT AGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGG TGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGC CGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCG ACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAA CCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAA CGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTT CTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTT CACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTA ACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTG CAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCA GCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTG AGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATT GGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCC GCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCAT TGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACG CGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCG CTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAG GCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGC CCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTG GTGTCAAAAATAA

TABLE 56 Pfnr3-lacZ construct Sequences Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 252) GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcact atcgtcgtccggccttttcctctcttactctgctacgtacatctatttctataaat ccgttcaatttgtctgttttttgcacaaacatgaaatatcagacaattccgtgact taagaaaatttatacaaatcagcaatataccccttaaggagtatataaaggtgaat ttgatttacatcaataagcggggttgctgaatcgttaaGGATCC ctctagaaataa ttttgtttaactttaagaaggagatatacat ATG ACTATGATTACGGATTCTCTGG CCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGC CTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA TCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTC CGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGAT ACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACAC CAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGA CAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAG ACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCG CTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCAT TTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGC AGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTC GTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTA ATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTG CGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAG CGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATC GCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCG AATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGC AGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGC TGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTG CATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCA GAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACA CGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCAC GGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAG CGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCT GGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGG ATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACAC CACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCT TCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATG CGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTT CGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCT GGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCG GCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGG TCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAAC AGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATAC CTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAA GCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGA TTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTA CGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTG GCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACG CCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAG CGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGA AAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACG ACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGG AAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATAC ACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAA CCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATC AATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGAC CTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAG AAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTG TCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGAC GCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCA GCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCG GAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGA CTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATT ACCAGTTGGTCTGGTGTCAAAAATAA

TABLE 57 Pfnr4-lacZ construct Sequences Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 253) GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatgg ctcatgcatgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcact cgacaggagtatttatattgcgcccGGATCC ctctagaaataattttgtttaactt taagaaggagatatacat ATG ACTATGATTACGGATTCTCTGGCCGTCGTATTACA ACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATC CCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGC GGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCC CCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTAT CCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTC GCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTT TTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTAC GGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGG AGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAG ATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAA CCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAG CCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGC GGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCT TTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACG CCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTG CAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGAC GTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCC GTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCA TGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAAC GCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCG CTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAA TGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACG CGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAA TGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCG ATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGAT ATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCC GAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCC TTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGG CAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGA TCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTG ATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCC GACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCA GTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATA GCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGC GGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGA ACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAAC CAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGT CTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACT GACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTA ACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTG ACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAG TGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCC ATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCG GTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAG CCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTG CGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCG CAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGA CCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATA CCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAAT TATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCA ACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACAT GGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCG TCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTG GTGTCAAAAATAA

TABLE 58 Pfnrs-lacZ construct Sequences Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 254) GGTACCagttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgt aacaaaagcaatttttccggctgtctgtatacaaaaacgccgtaaagtttgagcgaagt caataaactctctacccattcagggcaatatctctcttGGATCC ctctagaaataattt tgtttaactttaagaaggagatatacat ATG CTATGATTACGGATTCTCTGGCCGTCGT ATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCAC ATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGT GCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAA ACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATCCCATTACG GTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAA TATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACT CGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTG CCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGAT GGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCG GCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAA GTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGAT GTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGC AGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTAT GCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAAT CCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAG CAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTG AACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGG TCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACT TTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGAC CGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAAT GAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGA TGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCA GGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCG CCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGA TGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAA AAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGC GATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCC GTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAA AACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCA GTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAG CAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACC AGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGA TGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGT TGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTA CGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCA GCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCC CTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAA TTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCT GACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTG AAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTAC CAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGAT TACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCT ACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGAT ACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGT AAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTT TTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAA AACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGA CTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCC ATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATT GGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCG CTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Example 35 Increasing in vitro Butyrate and Acetate Production in Engineered Nissle

E. coli generates high levels of acetate as an end product of fermentation. In order to improve acetate production while also maintaining high levels butyrate production, deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and 1 dh (lactate dehydrogenase) were generated to prevent or reduce metabolic flux through pathways which do not result in acetate or butyrate production (see, e.g., FIG. 14). For this study, Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes were used. Additionally, for this study media M9 media containing 50 mM MOPS with 0.5% glucose was comparedto media containing 0.5/% glucuronic acid, as glucuronic acid better mimics available carbon sources in the gut.

Briefly, bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10⁸ CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose or 0.5% glucuronic acid in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein in Example 18 and Example 21.

As seen in FIG. 14A and FIG. 14B, both integrated strains made similar amounts of acetate, and FNRS-ter-pbt-buk butyrate cassettes produced slightly more butyrate. Deletions in adhE and ldhA have similar effects on butyrate and acetate production. Acetate production was much greater in media containing 0.5% glucuronic acid.

In alternate embodiments, frd (fumarate reductase) is deleted to assess the effect of the deletion on acetate and butyrate production.

Example 36 Generation of Indole Propionic Acid Strain and In Vitro indole Production

To facilitate inducible production of indole propionic acid (IPA) in Escherichia coli Nissle, 6 genes allowing the production of indole propionic acid from tryptophan, as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 under a tet inducible promoter. In other embodiments, the IPA synthesis cassette is put under the control of an FNR, RNS or ROS promoter, e.g., described herein, or other promoter induced by conditions in the healthy or diseased gut, e.g., inflammatory conditions. For efficient translation of IPA synthesis genes, each synthetic gene in the cassette is separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

The IPA synthesis cassette comprises TrpDH (tryptophan dehydrogenase from Nostoc punctiforme NIES-2108), FldH1/F1dH2 (indole-3-lactate dehydrogenase from Clostridium sporogenes), FldA (indole-3-propionyl-CoA: indole-3-lactate CoA transferase from Clostridium sporogenes), FldBC (indole-3-lactate dehydratase from Clostridium sporogenes), FldD (indole-3-acrylyl-CoA reductase from Clostridium sporogenes), and Acul (acrylyl-CoA reductase from Rhodobacter sphaeroides).

The tet inducible IPA construct described above is transformed into E. coli Nissle as described herein and production of IPA is assessed. In certain embodiments, E. coli Nissle strains containing the IPA synthesis cassette described further comprise a tryptophan synthesis cassette. In certain embodiments, the strains comprise a feedback resistant version of AroG and TrpE to achieve greater Trp production. In certain embodiments, additionally, the tnaA gene (tryptophanase converting Trp into indole) is deleted.

All incubations are performed at 37° C. LB-grown overnight cultures of E. coli Nissle transformed with the IPA biosynthesis construct alone or in combination with a tryptophan biosynthesis construct and feedback resistant AroG and TrpE are subcultured 1:100 into 10 mL of M9 minimal medium containing 0.5% glucose and grown shaking (200 rpm) for 2 h, at which time anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of the IPA biosynthesis and tryptophan biosynthesis constructs. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points (e.g., 0 up to 24 hours) by LC-MS to assess levels of IPA.

Production of IPA is also assessed in E. coli Nissle strains containing the IPA and tryptophan cassettes both driven by an RNS promoter e.g., a nsrR-norB-IPA biosynthesis construct) in order to assess nitrogen dependent induction of IPA production. Overnight bacterial cultures are diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) was added to cultures at a final concentration of 0.3 mM to induce expression from plasmid. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points (0 up to 24 hours) to assess IPA levels.

In alternate embodiments, production of IPA is also assessed in E. coli Nissle strains containing the IPA and tryptophan cassettes both driven by the low oxygen inducible FNR promoter, e.g., FNRS, or the reactive oxygen regulated OxyS promoter.

Example 37 Synthesis of Constructs for Synthesis of Tryptophan, Tryptamine, and Other Indole Metabolites

Various constructs were synthesized, and cloned into vector pBR322 for transformation of E. coli . In some embodiments, the constructs encoding the effector molecules are integrated into the genome according to methods described herein, e.g., Example 2.

TABLE 59 Sequences Description Sequence fbrAroG (RBS and Ctctagaaataattttgtttaactttaagaaggagatatacat leader region atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg underlined) cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga SEQ ID NO: 255 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa fbrAroG atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg SEQ ID NO: 256 cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa fbrAroG-serA Ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacg (RBS and leader atttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattcccc region underlined; gctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcct SerA starts after gaaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtc second RBS) gcggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagct SEQ ID NO: 257 ggaaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggc tgattaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgccc gcaaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggata tgatcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtacca ccgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaa atggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgc actgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca gtaaaagcgcgtcgcgggtaaTACT taagaaggagatatacatatggcaaaggtatcgctggagaaagacaagattaagtttctg ctggtagaaggcgtgcaccaaaaggcgctggaaagccttcgtgcagctggttacacca acatcgaatttcacaaaggcgcgctggatgatgaacaattaaaagaatccatccgcgat gcccacttcatcggcctgcgatcccgtacccatctgactgaagacgtgatcaacgccgc agaaaaactggtcgctattggctgtttctgtatcggaacaaatcaggttgatctggatgcg gcggcaaagcgcgggatcccggtatttaacgcaccgttctcaaatacgcgctctgttgc ggagctggtgattggcgaactgctgctgctattgcgcggcgtgccagaagccaatgcta aagcgcatcgtggcgtgtggaacaaactggcggcgggttcttttgaagcgcgcggcaa aaagctgggtatcatcggctacggtcatattggtacgcaattgggcattctggctgaatcg ctgggaatgtatgtttacttttatgatattgaaaacaaactgccgctgggcaacgccactca ggtacagcatctttctgacctgctgaatatgagcgatgtggtgagtctgcatgtaccagag aatccgtccaccaaaaatatgatgggcgcgaaagagatttcgctaatgaagcccggctc gctgctgattaatgcttcgcgcggtactgtggtggatattccagcgctgtgtgacgcgctg gcgagcaaacatctggcgggggcggcaatcgacgtattcccgacggaaccggcgac caatagcgatccatttacctctccgctgtgtgaattcgacaatgtccttctgacgccacaca ttggcggttcgactcaggaagcgcaggagaatatcggcttggaagttgcgggtaaattg atcaagtattctgacaatggctcaacgctctctgcggtgaacttcccggaagtctcgctgc cactgcacggtgggcgtcgtctgatgcacatccacgaaaaccgtccgggcgtgctaact gcgctcaacaaaatttttgccgagcagggcgtcaacatcgccgcgcaatatctacaaact tccgcccagatgggttatgtagttattgatattgaagccgacgaagacgttgccgaaaaa gcgctgcaggcaatgaaagctattccgggtaccattcgcgcccgtctgctgtactaa SerA atggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaaggcgtgca SEQ ID NO: 258 ccaaaaggcgctggaaagccttcgtgcagctggttacaccaacatcgaatttcacaaag gcgcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctg cgatcccgtacccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctat tggctgtttctgtatcggaacaaatcaggttgatctggatgcggcggcaaagcgcgggat cccggtatttaacgcaccgttctcaaatacgcgctctgttgcggagctggtgattggcga actgctgctgctattgcgcggcgtgccagaagccaatgctaaagcgcatcgtggcgtgt ggaacaaactggcggcgggttcttttgaagcgcgcggcaaaaagctgggtatcatcgg ctacggtcatattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttactt ttatgatattgaaaacaaactgccgctgggcaacgccactcaggtacagcatctttctgac ctgctgaatatgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaat atgatgggcgcgaaagagatttcgctaatgaagcccggctcgctgctgattaatgcttcg cgcggtactgtggtggatattccagcgctgtgtgacgcgctggcgagcaaacatctggc gggggcggcaatcgacgtattcccgacggaaccggcgaccaatagcgatccatttacc tctccgctgtgtgaattcgacaatgtccttctgacgccacacattggcggttcgactcagg aagcgcaggagaatatcggcttggaagttgcgggtaaattgatcaagtattctgacaatg gctcaacgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgt cgtctgatgcacatccacgaaaaccgtccgggcgtgctaactgcgctcaacaaaattttt gccgagcagggcgtcaacatcgccgcgcaatatctacaaacttccgcccagatgggtt atgtagttattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatg aaagctattccgggtaccattcgcgcccgtctgctgtactaa fbrAroG-Tdc (tdc ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacga from C. roseus); tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg RBS and leader ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg region underlined aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc SEQ ID NO: 259 ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca gtaaaagcgcgtcgcgggtaaTACTtaagaaggagatatacatATGGGTTC TATTGACTCGACGAATGTGGCCATGTCTAATTCTCCT GTTGGCGAGTTTAAGCCCCTTGAAGCAGAAGAGTTCC GTAAACAGGCACACCGCATGGTGGATTTTATTGCGGA TTATTACAAGAACGTAGAAACATACCCGGTCCTTTCC GAGGTTGAACCCGGCTATCTGCGCAAACGTATTCCCG AAACCGCACCATACCTGCCGGAGCCACTTGATGATAT TATGAAGGATATTCAAAAGGACATTATCCCCGGAAT GACGAACTGGATGTCCCCGAACTTTTACGCCTTCTTC CCGGCCACAGTTAGCTCAGCAGCTTTCTTGGGGGAAA TGCTTTCAACGGCCCTTAACAGCGTAGGATTTACCTG GGTCAGTTCCCCGGCAGCGACTGAATTAGAGATGATC GTTATGGATTGGCTTGCGCAAATTTTGAAACTTCCAA AAAGCTTTATGTTCTCCGGAACCGGGGGTGGTGTCAT CCAAAACACTACGTCAGAGTCGATCTTGTGCACTATT ATCGCGGCCCGTGAACGCGCCTTGGAAAAATTGGGC CCTGATTCAATTGGTAAGCTTGTCTGCTATGGGTCCG ATCAAACGCACACAATGTTTCCGAAAACCTGTAAGTT AGCAGGAATTTATCCGAATAATATCCGCCTTATCCCT ACCACGGTAGAAACCGACTTTGGCATCTCACCGCAG GTACTTCGCAAGATGGTCGAAGACGACGTCGCTGCG GGGTACGTTCCCTTATTTTTGTGTGCCACCTTGGGAA CGACATCAACTACGGCAACAGATCCTGTAGATTCGCT GTCCGAAATCGCAAACGAGTTTGGTATCTGGATTCAT GTCGACGCCGCATATGCTGGATCGGCTTGCATCTGCC CAGAATTTCGTCACTACCTTGATGGCATCGAACGTGT GGATTCCTTATCGCTGTCTCCCCACAAATGGCTTTTA CCACGTAATTTTTCGCTTGTATGCTTTCGCTTGAAACC GGATGTATCTAGTTTACATGTCGAGGAGGTCAACAAG AAGTTGTTGGATATGCTTAACTCCACCGGTCGCGTAT ATATGACGCATACAATTGTTGGCGGAATCTATATGTT ACGTTTGGCTGTAGGTAGCAGCTTGACAGAGGAACA TCACGTGCGCCGCGTTTGGGACTTGATCCAGAAGCTT ACGGACGACCTGCTTAAAGAGGCGTGA Tdc (tdc from ATGGGTTCTATTGACTCGACGAATGTGGCCATGTCTA C. roseus) ATTCTCCTGTTGGCGAGTTTAAGCCCCTTGAAGCAGA SEQ ID NO: 260 AGAGTTCCGTAAACAGGCACACCGCATGGTGGATTTT ATTGCGGATTATTACAAGAACGTAGAAACATACCCG GTCCTTTCCGAGGTTGAACCCGGCTATCTGCGCAAAC GTATTCCCGAAACCGCACCATACCTGCCGGAGCCACT TGATGATATTATGAAGGATATTCAAAAGGACATTATC CCCGGAATGACGAACTGGATGTCCCCGAACTTTTACG CCTTCTTCCCGGCCACAGTTAGCTCAGCAGCTTTCTTG GGGGAAATGCTTTCAACGGCCCTTAACAGCGTAGGA TTTACCTGGGTCAGTTCCCCGGCAGCGACTGAATTAG AGATGATCGTTATGGATTGGCTTGCGCAAATTTTGAA ACTTCCAAAAAGCTTTATGTTCTCCGGAACCGGGGGT GGTGTCATCCAAAACACTACGTCAGAGTCGATCTTGT GCACTATTATCGCGGCCCGTGAACGCGCCTTGGAAAA ATTGGGCCCTGATTCAATTGGTAAGCTTGTCTGCTAT GGGTCCGATCAAACGCACACAATGTTTCCGAAAACCT GTAAGTTAGCAGGAATTTATCCGAATAATATCCGCCT TATCCCTACCACGGTAGAAACCGACTTTGGCATCTCA CCGCAGGTACTTCGCAAGATGGTCGAAGACGACGTC GCTGCGGGGTACGTTCCCTTATTTTTGTGTGCCACCTT GGGAACGACATCAACTACGGCAACAGATCCTGTAGA TTCGCTGTCCGAAATCGCAAACGAGTTTGGTATCTGG ATTCATGTCGACGCCGCATATGCTGGATCGGCTTGCA TCTGCCCAGAATTTCGTCACTACCTTGATGGCATCGA ACGTGTGGATTCCTTATCGCTGTCTCCCCACAAATGG CTTTTAGCATATCTGGATTGCACGTGCTTGTGGGTAA AACAACCTCACCTGCTGCTTCGCGCTTTAACGACTAA TCCCGAATACTTGAAGAATAAACAGAGTGATTTAGAT AAGGTCGTGGATTTTAAGAACTGGCAGATCGCAACA GGACGTAAGTTCCGCTCTTTAAAACTTTGGTTAATTC TGCGTTCCTACGGGGTAGTTAACCTGCAAAGTCATAT CCGTAGTGATGTAGCGATGGGGAAGATGTTTGAGGA ATGGGTCCGTTCCGATAGCCGCTTTGAAATCGTCGTG CCACGTAATTTTTCGCTTGTATGCTTTCGCTTGAAACC GGATGTATCTAGTTTACATGTCGAGGAGGTCAACAAG AAGTTGTTGGATATGCTTAACTCCACCGGTCGCGTAT ATATGACGCATACAATTGTTGGCGGAATCTATATGTT ACGTTTGGCTGTAGGTAGCAGCTTGACAGAGGAACA TCACGTGCGCCGCGTTTGGGACTTGATCCAGAAGCTT ACGGACGACCTGCTTAAAGAGGCGTGA fbrAroG-Tdc (tdc ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacga from Clostridium tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg sporogenes); RBS ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg and leader region aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc underlined ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg SEQ ID NO: 261 aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca gtaaaagcgcgtcgcgggtaaTACTtaagaaggagatatacatATGAAATT TTGGCGCAAGTATACGCAACAGGAGATGGATGAGAA AATCACAGAATCGCTTGAGAAGACATTAAATTACGA TAACACGAAAACCATCGGCATCCCAGGTACTAAGCT GGATGATACTGTATTTTATGACGATCACTCCTTCGTT AAGCACTCTCCCTATTTACGTACGTTCATCCAAAACC CTAATCACATTGGTTGTCACACGTACGATAAAGCAGA CATCTTGTTTGGCGGCACGTTTGACATCGAACGCGAA CTGATTCAGCTTTTGGCCATCGATGTCTTAAACGGAA ATGATGAGGAATTCGATGGATATGTGACACAGGGGG GAACCGAGGCGAATATTCAGGCAATGTGGGTTTATC GTAACTATTTCAAAAAAGAACGTAAAGCAAAACATG AGGAAATCGCAATCATCACGAGCGCGGATACCCATT ACAGTGCATATAAGGGGAGCGACTTGCTGAACATTG ATATTATCAAGGTCCCAGTAGACTTCTATTCGCGTAA GATCCAGGAGAACACGTTAGACTCGATTGTCAAGGA GGCGAAGGAAATTGGAAAGAAGTACTTCATTGTCAT CTCAAACATGGGTACGACTATGTTTGGCAGTGTAGAC GACCCTGATCTTTATGCTAACATTTTTGATAAGTATA ACTTAGAATACAAAATCCACGTCGATGGAGCTTTTGG GGGTTTCATTTATCCTATCGATAATAAGGAGTGCAAA ACAGATTTCTCGAACAAGAACGTCTCATCCATCACGC TTGACGGTCACAAAATGCTTCAAGCCCCCTATGGGAC TGGTATCTTCGTGTCACGTAAGAACTTGATCCATAAC ACCCTGACAAAGGAAGCAACGTATATTGAAAACCTG GACGTTACCCTGAGTGGGTCCCGCTCCGGATCCAACG CCGTTGCGATCTGGATGGTTTTAGCCTCTTATGGCCC CTACGGGTGGATGGAGAAGATTAACAAGTTGCGCAA TCGCACTAAGTGGCTTTGCAAGCAGCTTAACGACATG CGCATCAAATACTATAAGGAGGATAGCATGAATATC GTCACGATTGAAGAGCAATACGTAAATAAAGAGATT GCAGAGAAATACTTCCTTGTGCCTGAAGTACACAATC CTACCAACAATTGGTACAAGATTGTAGTCATGGAACA TGTTGAACTTGACATCTTGAACTCCCTTGTTTATGATT TACGTAAATTCAACAAGGAGCACCTGAAGGCAATGT GA Tdc (tdc from ATGAAATTTTGGCGCAAGTATACGCAACAGGAGATG Clostridium GATGAGAAAATCACAGAATCGCTTGAGAAGACATTA sporogenes) AATTACGATAACACGAAAACCATCGGCATCCCAGGT SEQ ID NO: 262 ACTAAGCTGGATGATACTGTATTTTATGACGATCACT CCTTCGTTAAGCACTCTCCCTATTTACGTACGTTCATC CAAAACCCTAATCACATTGGTTGTCACACGTACGATA AAGCAGACATCTTGTTTGGCGGCACGTTTGACATCGA ACGCGAACTGATTCAGCTTTTGGCCATCGATGTCTTA AACGGAAATGATGAGGAATTCGATGGATATGTGACA CAGGGGGGAACCGAGGCGAATATTCAGGCAATGTGG GTTTATCGTAACTATTTCAAAAAAGAACGTAAAGCAA AACATGAGGAAATCGCAATCATCACGAGCGCGGATA CCCATTACAGTGCATATAAGGGGAGCGACTTGCTGA ACATTGATATTATCAAGGTCCCAGTAGACTTCTATTC GCGTAAGATCCAGGAGAACACGTTAGACTCGATTGT CAAGGAGGCGAAGGAAATTGGAAAGAAGTACTTCAT TGTCATCTCAAACATGGGTACGACTATGTTTGGCAGT GTAGACGACCCTGATCTTTATGCTAACATTTTTGATA AGTATAACTTAGAATACAAAATCCACGTCGATGGAG CTTTTGGGGGTTTCATTTATCCTATCGATAATAAGGA GTGCAAAACAGATTTCTCGAACAAGAACGTCTCATCC ATCACGCTTGACGGTCACAAAATGCTTCAAGCCCCCT ATGGGACTGGTATCTTCGTGTCACGTAAGAACTTGAT CCATAACACCCTGACAAAGGAAGCAACGTATATTGA AAACCTGGACGTTACCCTGAGTGGGTCCCGCTCCGGA TCCAACGCCGTTGCGATCTGGATGGTTTTAGCCTCTT ATGGCCCCTACGGGTGGATGGAGAAGATTAACAAGT TGCGCAATCGCACTAAGTGGCTTTGCAAGCAGCTTAA CGACATGCGCATCAAATACTATAAGGAGGATAGCAT GAATATCGTCACGATTGAAGAGCAATACGTAAATAA AGAGATTGCAGAGAAATACTTCCTTGTGCCTGAAGTA CACAATCCTACCAACAATTGGTACAAGATTGTAGTCA TGGAACATGTTGAACTTGACATCTTGAACTCCCTTGT TTATGATTTACGTAAATTCAACAAGGAGCACCTGAAG GCAATGTGA fbrArG-trpDH- Ctctagaaataattttgtttaactttaagaaggagatatacat ipdC-iad1 (RBS atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg and leader region cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga underlined) aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca SEQ ID NO: 263 tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACTtaagaaggaga tatacatATGCTGTTATTCGAGACTGTGCGTGAAATGGGT CATGAGCAAGTCCTTTTCTGTCATAGCAAGAATCCCG AGATCAAGGCAATTATCGCAATCCACGATACCACCTT AGGACCGGCTATGGGCGCAACTCGTATCTTACCTTAT ATTAATGAGGAGGCTGCCCTGAAAGATGCATTACGTC TGTCCCGCGGAATGACTTACAAAGCAGCCTGCGCCA ATATTCCCGCCGGGGGCGGCAAAGCCGTCATCATCGC TAACCCCGAAAACAAGACCGATGACCTGTTACGCGC ATACGGCCGTTTCGTGGACAGCTTGAACGGCCGTTTC ATCACCGGGCAGGACGTTAACATTACGCCCGACGAC GTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAG GCGTCTCAGAAAAGTCGGGAGGGCCGGCACCTATCA CCTCTCTGGGAGTATTTTTAGGCATCAAAGCCGCTGT AGAGTCGCGTTGGCAGTCTAAACGCCTGGATGGCAT GAAAGTGGCGGTGCAAGGACTTGGGAACGTAGGAAA AAATCTTTGTCGCCATCTGCATGAACACGATGTACAA CTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGAGG AAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACC GACTGAAATCTATTCTTTAGATGTTGATATTTTTGCAC CGTGTGCACTTGGGGGTATTTTGAATAGCCATACCAT CCCGTTCTTACAAGCCTCAATCATCGCAGGAGCAGCG AATAACCAGCTGGAGAACGAGCAACTTCATTCGCAG ATGCTTGCGAAAAAGGGTATTCTTTACTCACCAGACT ACGTTATCAATGCAGGAGGACTTATCAATGTTTATAA CGAAATGATCGGATATGACGAGGAAAAAGCATTCAA ACAAGTTCATAACATCTACGATACGTTATTAGCGATT TTCGAAATTGCAAAAGAACAAGGTGTAACCACCAAC GACGCGGCCCGTCGTTTAGCAGAGGATCGTATCAAC AACTCCAAACGCTCAAAGAGTAAAGCGATTGCGGCG TGAAATGtaagaaggagatatacatATGCGTACACCCTACTGTG TCGCCGATTATCTTTTAGATCGTCTGACGGACTGCGG GGCCGATCACCTGTTTGGCGTACCGGGCGATTACAAC TTGCAGTTTCTGGACCACGTCATTGACTCACCAGATA TCTGCTGGGTAGGGTGTGCGAACGAGCTTAACGCGA GCTACGCTGCTGACGGATATGCGCGTTGTAAAGGCTT TGCTGCACTTCTTACTACCTTCGGGGTCGGTGAGTTA TCGGCGATGAACGGTATCGCAGGCTCGTACGCTGAG CACGTCCCGGTATTACACATTGTGGGAGCTCCGGGTA CCGCAGCTCAACAGCGCGGAGAACTGTTACACCACA CGCTGGGCGACGGAGAATTCCGCCACTTTTACCATAT GTCCGAGCCAATTACTGTAGCCCAGGCTGTACTTACA GAGCAAAATGCCTGTTACGAGATCGACCGTGTTTTGA CCACGATGCTTCGCGAGCGCCGTCCCGGGTATTTGAT GCTGCCAGCCGATGTTGCCAAAAAAGCTGCGACGCC CCCAGTGAATGCCCTGACGCATAAACAAGCTCATGCC GATTCCGCCTGTTTAAAGGCTTTTCGCGATGCAGCTG AAAATAAATTAGCCATGTCGAAACGCACCGCCTTGTT GGCGGACTTTCTGGTCCTGCGCCATGGCCTTAAACAC GCCCTTCAGAAATGGGTCAAAGAAGTCCCGATGGCC CACGCTACGATGCTTATGGGTAAGGGGATTTTTGATG AACGTCAAGCGGGATTTTATGGAACTTATTCCGGTTC GGCGAGTACGGGGGCGGTAAAGGAAGCGATTGAGGG AGCCGACACAGTTCTTTGCGTGGGGACACGTTTCACC GATACACTGACCGCTGGATTCACACACCAACTTACTC CGGCACAAACGATTGAGGTGCAACCCCATGCGGCTC GCGTGGGGGATGTATGGTTTACGGGCATTCCAATGAA TCAAGCCATTGAGACTCTTGTCGAGCTGTGCAAACAG CACGTCCACGCAGGACTGATGAGTTCGAGCTCTGGG GCGATTCCTTTTCCACAACCAGATGGTAGTTTAACTC AAGAAAACTTCTGGCGCACATTGCAAACCTTTATCCG CCCAGGTGATATCATCTTAGCAGACCAGGGTACTTCA GCCTTTGGAGCAATTGACCTGCGCTTACCAGCAGACG TGAACTTTATTGTGCAGCCGCTGTGGGGGTCTATTGG TTATACTTTAGCTGCGGCCTTCGGAGCGCAGACAGCG TGTCCAAACCGTCGTGTGATCGTATTGACAGGAGATG GAGCAGCGCAGTTGACCATTCAGGAGTTAGGCTCGA TGTTACGCGATAAGCAGCACCCCATTATCCTGGTCCT GAACAATGAGGGGTATACAGTTGAACGCGCCATTCA TGGTGCGGAACAACGCTACAATGACATCGCTTTATGG AATTGGACGCACATCCCCCAAGCCTTATCGTTAGATC CCCAATCGGAATGTTGGCGTGTGTCTGAAGCAGAGC AACTGGCTGATGTTCTGGAAAAAGTTGCTCATCATGA ACGCCTGTCGTTGATCGAGGTAATGTTGCCCAAGGCC GATATCCCTCCGTTACTGGGAGCCTTGACCAAGGCTT TAGAAGCCTGCAACAACGCTTAAAGGTtaagaaggagatata catATGCCCACCTTGAACTTGGACTTACCCAACGGTAT TAAGAGCACGATTCAGGCAGACCTTTTCATCAATAAT AAGTTTGTGCCGGCGCTTGATGGGAAAACGTTCGCAA CTATTAATCCGTCTACGGGGAAAGAGATCGGACAGG TGGCAGAGGCTTCGGCGAAGGATGTGGATCTTGCAG TTAAGGCCGCGCGTGAGGCGTTTGAAACTACTTGGGG GGAAAACACGCCAGGTGATGCTCGTGGCCGTTTACTG ATTAAGCTTGCTGAGTTGGTGGAAGCGAATATTGATG AGTTAGCGGCAATTGAATCACTGGACAATGGGAAAG CGTTCTCTATTGCTAAGTCATTCGACGTAGCTGCTGT GGCCGCAAACTTACGTTACTACGGCGGTTGGGCTGAT AAAAACCACGGTAAAGTCATGGAGGTAGACACAAAG CGCCTGAACTATACCCGCCACGAGCCGATCGGGGTTT GCGGACAAATCATTCCGTGGAATTTCCCGCTTTTGAT GTTTGCATGGAAGCTGGGTCCCGCTTTAGCCACAGGG AACACAATTGTGTTAAAGACTGCCGAGCAGACTCCCT TAAGTGCTATCAAGATGTGTGAATTAATCGTAGAAGC CGGCTTTCCGCCCGGAGTAGTTAATGTGATCTCGGGA TTCGGACCGGTGGCGGGGGCCGCGATCTCGCAACAC ATGGACATCGATAAGATTGCCTTTACAGGATCGACAT TGGTTGGCCGCAACATTATGAAGGCAGCTGCGTCGAC TAACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAA ATCCCCGAATATCATTTTCAAAGATGCCGACCTTGAC CAAGCTGTTCGCTGGAGCGCCTTCGGTATCATGTTTA ACCACGGACAATGCTGCTGCGCTGGATCGCGCGTATA TGTGGAAGAATCCATCTATGACGCCTTCATGGAAAAA ATGACTGCGCATTGTAAGGCGCTTCAAGTTGGAGATC CTTTCAGCGCGAACACCTTCCAAGGACCACAAGTCTC GCAGTTACAATACGACCGTATCATGGAATACATCGA ATCAGGGAAAAAAGATGCAAATCTTGCTTTAGGCGG CGTTCGCAAAGGGAATGAGGGGTATTTCATTGAGCC AACTATTTTTACAGACGTGCCGCACGACGCGAAGATT GCCAAAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGT CGAAATTTAAGGACGAAAAAGATCTGATCCGTATCG CAAATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTT TCCCGCGACATCAGCCGCGCGATCGAGACAGCACAC AAACTGAAAGCAGGCACGGTCTGGGTCAACTGCTAT AATCAGCTTATTCCGCAGGTGCCATTCGGAGGGTATA AGGCTTCCGGTATCGGCCGTGAGTTGGGGGAATATGC CTTGTCTAATTACACAAATATCAAGGCCGTCCACGTT AACCTTTCTCAACCGGCGCCCATTTGA trpDH ATGCTGTTATTCGAGACTGTGCGTGAAATGGGTCATG SEQ ID NO: 264 AGCAAGTCCTTTTCTGTCATAGCAAGAATCCCGAGAT CAAGGCAATTATCGCAATCCACGATACCACCTTAGGA CCGGCTATGGGCGCAACTCGTATCTTACCTTATATTA ATGAGGAGGCTGCCCTGAAAGATGCATTACGTCTGTC CCGCGGAATGACTTACAAAGCAGCCTGCGCCAATATT CCCGCCGGGGGCGGCAAAGCCGTCATCATCGCTAAC CCCGAAAACAAGACCGATGACCTGTTACGCGCATAC GGCCGTTTCGTGGACAGCTTGAACGGCCGTTTCATCA CCGGGCAGGACGTTAACATTACGCCCGACGACGTTC GCACTATTTCGCAGGAGACTAAGTACGTGGTAGGCGT CTCAGAAAAGTCGGGAGGGCCGGCACCTATCACCTC TCTGGGAGTATTTTTAGGCATCAAAGCCGCTGTAGAG TCGCGTTGGCAGTCTAAACGCCTGGATGGCATGAAA GTGGCGGTGCAAGGACTTGGGAACGTAGGAAAAAAT CTTTGTCGCCATCTGCATGAACACGATGTACAACTTT TTGTGTCTGATGTCGATCCAATCAAGGCCGAGGAAGT AAAACGCTTATTCGGGGCGACTGTTGTCGAACCGACT GAAATCTATTCTTTAGATGTTGATATTTTTGCACCGTG TGCACTTGGGGGTATTTTGAATAGCCATACCATCCCG TTCTTACAAGCCTCAATCATCGCAGGAGCAGCGAATA ACCAGCTGGAGAACGAGCAACTTCATTCGCAGATGC TTGCGAAAAAGGGTATTCTTTACTCACCAGACTACGT TATCAATGCAGGAGGACTTATCAATGTTTATAACGAA ATGATCGGATATGACGAGGAAAAAGCATTCAAACAA GTTCATAACATCTACGATACGTTATTAGCGATTTTCG AAATTGCAAAAGAACAAGGTGTAACCACCAACGACG CGGCCCGTCGTTTAGCAGAGGATCGTATCAACAACTC CAAACGCTCAAAGAGTAAAGCGATTGCGGCGTGA ipdC ATGCGTACACCCTACTGTGTCGCCGATTATCTTTTAG SEQ ID NO: 265 ATCGTCTGACGGACTGCGGGGCCGATCACCTGTTTGG CGTACCGGGCGATTACAACTTGCAGTTTCTGGACCAC GTCATTGACTCACCAGATATCTGCTGGGTAGGGTGTG CGAACGAGCTTAACGCGAGCTACGCTGCTGACGGAT ATGCGCGTTGTAAAGGCTTTGCTGCACTTCTTACTAC CTTCGGGGTCGGTGAGTTATCGGCGATGAACGGTATC GCAGGCTCGTACGCTGAGCACGTCCCGGTATTACACA TTGTGGGAGCTCCGGGTACCGCAGCTCAACAGCGCG GAGAACTGTTACACCACACGCTGGGCGACGGAGAAT TCCGCCACTTTTACCATATGTCCGAGCCAATTACTGT AGCCCAGGCTGTACTTACAGAGCAAAATGCCTGTTAC GAGATCGACCGTGTTTTGACCACGATGCTTCGCGAGC GCCGTCCCGGGTATTTGATGCTGCCAGCCGATGTTGC CAAAAAAGCTGCGACGCCCCCAGTGAATGCCCTGAC GCATAAACAAGCTCATGCCGATTCCGCCTGTTTAAAG GCTTTTCGCGATGCAGCTGAAAATAAATTAGCCATGT CGAAACGCACCGCCTTGTTGGCGGACTTTCTGGTCCT GCGCCATGGCCTTAAACACGCCCTTCAGAAATGGGTC AAAGAAGTCCCGATGGCCCACGCTACGATGCTTATG GGTAAGGGGATTTTTGATGAACGTCAAGCGGGATTTT ATGGAACTTATTCCGGTTCGGCGAGTACGGGGGCGGT AAAGGAAGCGATTGAGGGAGCCGACACAGTTCTTTG CGTGGGGACACGTTTCACCGATACACTGACCGCTGGA TTCACACACCAACTTACTCCGGCACAAACGATTGAGG TGCAACCCCATGCGGCTCGCGTGGGGGATGTATGGTT TACGGGCATTCCAATGAATCAAGCCATTGAGACTCTT GTCGAGCTGTGCAAACAGCACGTCCACGCAGGACTG ATGAGTTCGAGCTCTGGGGCGATTCCTTTTCCACAAC CAGATGGTAGTTTAACTCAAGAAAACTTCTGGCGCAC ATTGCAAACCTTTATCCGCCCAGGTGATATCATCTTA GCAGACCAGGGTACTTCAGCCTTTGGAGCAATTGACC TGCGCTTACCAGCAGACGTGAACTTTATTGTGCAGCC GCTGTGGGGGTCTATTGGTTATACTTTAGCTGCGGCC TTCGGAGCGCAGACAGCGTGTCCAAACCGTCGTGTG ATCGTATTGACAGGAGATGGAGCAGCGCAGTTGACC ATTCAGGAGTTAGGCTCGATGTTACGCGATAAGCAGC ACCCCATTATCCTGGTCCTGAACAATGAGGGGTATAC AGTTGAACGCGCCATTCATGGTGCGGAACAACGCTA CAATGACATCGCTTTATGGAATTGGACGCACATCCCC CAAGCCTTATCGTTAGATCCCCAATCGGAATGTTGGC GTGTGTCTGAAGCAGAGCAACTGGCTGATGTTCTGGA AAAAGTTGCTCATCATGAACGCCTGTCGTTGATCGAG GTAATGTTGCCCAAGGCCGATATCCCTCCGTTACTGG GAGCCTTGACCAAGGCTTTAGAAGCCTGCAACAACG CTTAA Iad1 ATGCCCACCTTGAACTTGGACTTACCCAACGGTATTA SEQ ID NO: 266 AGAGCACGATTCAGGCAGACCTTTTCATCAATAATAA GTTTGTGCCGGCGCTTGATGGGAAAACGTTCGCAACT ATTAATCCGTCTACGGGGAAAGAGATCGGACAGGTG GCAGAGGCTTCGGCGAAGGATGTGGATCTTGCAGTT AAGGCCGCGCGTGAGGCGTTTGAAACTACTTGGGGG GAAAACACGCCAGGTGATGCTCGTGGCCGTTTACTGA TTAAGCTTGCTGAGTTGGTGGAAGCGAATATTGATGA GTTAGCGGCAATTGAATCACTGGACAATGGGAAAGC GTTCTCTATTGCTAAGTCATTCGACGTAGCTGCTGTG GCCGCAAACTTACGTTACTACGGCGGTTGGGCTGATA AAAACCACGGTAAAGTCATGGAGGTAGACACAAAGC GCCTGAACTATACCCGCCACGAGCCGATCGGGGTTTG CGGACAAATCATTCCGTGGAATTTCCCGCTTTTGATG TTTGCATGGAAGCTGGGTCCCGCTTTAGCCACAGGGA ACACAATTGTGTTAAAGACTGCCGAGCAGACTCCCTT AAGTGCTATCAAGATGTGTGAATTAATCGTAGAAGCC GGCTTTCCGCCCGGAGTAGTTAATGTGATCTCGGGAT TCGGACCGGTGGCGGGGGCCGCGATCTCGCAACACA TGGACATCGATAAGATTGCCTTTACAGGATCGACATT GGTTGGCCGCAACATTATGAAGGCAGCTGCGTCGACT AACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAAA TCCCCGAATATCATTTTCAAAGATGCCGACCTTGACC AAGCTGTTCGCTGGAGCGCCTTCGGTATCATGTTTAA CCACGGACAATGCTGCTGCGCTGGATCGCGCGTATAT GTGGAAGAATCCATCTATGACGCCTTCATGGAAAAA ATGACTGCGCATTGTAAGGCGCTTCAAGTTGGAGATC CTTTCAGCGCGAACACCTTCCAAGGACCACAAGTCTC GCAGTTACAATACGACCGTATCATGGAATACATCGA ATCAGGGAAAAAAGATGCAAATCTTGCTTTAGGCGG CGTTCGCAAAGGGAATGAGGGGTATTTCATTGAGCC AACTATTTTTACAGACGTGCCGCACGACGCGAAGATT GCCAAAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGT CGAAATTTAAGGACGAAAAAGATCTGATCCGTATCG CAAATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTT TCCCGCGACATCAGCCGCGCGATCGAGACAGCACAC AAACTGAAAGCAGGCACGGTCTGGGTCAACTGCTAT AATCAGCTTATTCCGCAGGTGCCATTCGGAGGGTATA AGGCTTCCGGTATCGGCCGTGAGTTGGGGGAATATGC CTTGTCTAATTACACAAATATCAAGGCCGTCCACGTT AACCTTTCTCAACCGGCGCCCATTTGA TrpEDCBA (RBS Ctctagaaataattttgtttaactttaagaaggagatatacat and leader region atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa underlined) cccgactgcgctattcaccagagtgtggggatcgtccggcaacgctgctgctggaatc SEQ ID NO: 267 cgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc gcattacagcattaagtgacactgtcacaatccaggcgctaccggcaatggagaagcc ctgagacactactggataacgccagcctgcgggtgtggaaaatgaacaatcaccaaac tgccgcgtactgcgcacccgcctgtcagtccactgctggatgaagacgcccgcttatgc tcccatcggtattgacgctaccgcttattacagaatctgagaatgtaccgaaggaagaa cgagaagcaatgacttcggcggcctgactcttatgaccagtggcgggatttgaaaattt accgcaactgtcagcggaaaatagctgccctgatactgatttatctcgctgaaacgctga tggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgatgctccgaatg aagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccga agccgcgccgccgctgccggtggtaccgtgccgcatatgcgagtgaatgtaaccaga gcgatgaagagacggtggtgtagtgcgatgagcaaaaagcgattcgcgccggagaa attaccaggtggtgccatctcgccgtactctctgccctgcccgtcaccgctggcagccta ttacgtgctgaaaaagagtaatcccagcccgtacatgatatatgcaggataatgatacac cctgtaggcgcgtcgccggaaagacgctcaagtatgacgccaccagccgccagattg agatttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggacgctggac agagacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctactga acatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggca gccgctacgtcgccgatctcaccaaagagaccgttactcttacgtgatgcacctagtctc ccgcgagaggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtat gaatatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaag cagaaggtcgtcgacgcggcagctacggcggcgcggtaggttatataccgcgcatgg cgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccg tgcaagccggtgctggcgtagtccagattctgaccgcagtcggaagccgacgaaactc gtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagac gactaatggctgacattctgctgctcgataatatcgactcattacgtacaacctggcagat cagagcgcagcaatggtcataacgtggtgatttaccgcaaccatattccggcgcagacc ttaattgaacgcctggcgacgatgagcaatccggtgctgatgctactcctggccccggt gtgccgagcgaagccggagtatgccggaactcctcacccgcttgcgtggcaagctgc caattattggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcgg tcaggcgggcgaaattcacacggtaaagcgtcgagcattgaacatgacggtcaggcg atgatgccggattaacaaacccgctgccagtggcgcgttatcactcgctggaggcagt aacattccggccggataaccatcaacgcccatataatggcatggtgatggcggtgcgtc acgatgcagatcgcgtagtggattccagaccatccggaatccattcttactacccaggg cgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaa cacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaa agccaccagctgattcagcggtggtacgtggcgagctgaagccggaacaactggcgg cggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccggggcag caaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgatgccgat atcgtcggtactggcggtgacggcagcaacagcatcaatatactaccgccagtgcgat gtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagt aaatccggctcgtcggatctgctggcggcgacggtattaatcagatatgaacgccgata aatcgcgccaggcgctggatgagttaggcgtctgatcctcatgcgccgaagtatcaca ccggattccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaac gtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtc cggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggc agtggtgcacagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccg aactacatgacggcgaaattaagagctatcaattgaccgctgaagattttggcctgacac cctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaac acgcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtc gccatgttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttctt gaggtactgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggt aaatgatgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacc cgcaaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcga catttttatgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgt cgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgccgccatttata aacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatt tcctccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgat ccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcagta ctggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgt gctgaccgaagtcagtaatgaagaggaactggagcgcgccattgcattgggggcaaa ggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccg cgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaat acttacgctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcggcgtt gatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaag tatgtggcctgacacgtgggcaagatgctaaagcagcttatgacgcgggcgcgatttac ggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgcaggaa gtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatg tggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagat cagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggc tttaagtgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcg acaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatggtcaatcg cttggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcac aaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaag acgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaat gacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctga tgcctgctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttc aggctcagttcaacgacctgctgaaaaactatgccgggcgtccaaccgcgctgaccaa atgccagaacattacagccgggacgaacaccacgctgtatctgaagcgcgaagatttg ctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagc ggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtc ggcccttgccagcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacg ttgaacgccagtcgcccaacgttttccggatgcgcttaatgggtgcggaagtgatcccg gtacatagcggttccgcgaccctgaaagatgcctgtaatgaggcgctacgcgactggtc cggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatccttacc cgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctg gaaagagaaggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgc catcggtatgtttgcagatttcatcaacgaaaccgacgtcggcctgattggtgtggagcct ggcggccacggtatcgaaactggcgagcacggcgcaccgttaaaacatggtcgcgtg ggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaattgaaga gtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctca acagcactggacgcgctgattacgtgtctattaccgacgatgaagccctggaagccttta aaacgctttgcctgcatgaagggatcatcccggcgctggaatcctcccacgccctggcc catgcgctgaaaatgatgcgcgaaaatccggaaaaagagcagctactggtggttaacct ttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagcacgagggga aatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgc attcgttcctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgata cgctaattgaagccggtgctgacgcgctggagttaggcatccccttctccgacccactg gcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcgggagtaaccc cggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatc ggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtg cgagaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcg cccttccgccaggccgcgttgcgtcataatgtcgcacctatctttatttgcccgccgaatg ccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcg cgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttg cgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccg gatcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccat cgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaag cttttgtacaaccgatgaaagcggcgacgcgcagtta trpE atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa SEQ ID NO: 268 cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaatc cgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagcc ctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaac tgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgc tccctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaa cgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaattt accgcaactgtcagcggaaaatagctgccctgatttctgatttatctcgctgaaacgctga tggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatg aagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccga agccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccaga gcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaa attttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagccta ttacgtgctgaaaaagagtaatcccagcccgtacatgattttatgcaggataatgatttcac cctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattg agatttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggac agagacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctga acatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggca gccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctc ccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtat gaatatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaag cagaaggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatgg cgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccg tgcaagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaaactc gtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagac gttcta trpD atggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagtt SEQ ID NO: 269 gcgcagcaatggtcataacgtggtgatttaccgcaaccatattccggcgcagaccttaat tgaacgcctggcgacgatgagcaatccggtgctgatgctttctcctggccccggtgtgc cgagcgaagccggttgtatgccggaactcctcacccgcttgcgtggcaagctgccaatt attggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtcag gcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtt tgccggattaacaaacccgctgccagtggcgcgttatcactcgctggttggcagtaacat tccggccggtttaaccatcaacgcccattttaatggcatggtgatggcggtgcgtcacga tgcagatcgcgtttgtggattccagttccatccggaatccattcttactacccagggcgctc gcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacg ctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagcc accagctgttttcagcggtggtacgtggcgagctgaagccggaacaactggcggcggc gctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccggggcagcaac cgcgctactggaaaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgt cggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgc cgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatc cggctcgtcggatctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcg cgccaggcgctggatgagttaggcgtctgtttcctctttgcgccgaagtatcacaccgga ttccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctg ggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtccggaa ctggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggt gcacagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactac atgacggcgaaattaagagctatcaattgaccgctgaagattttggcctgacaccctacc accaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacacgctt gttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccat gttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggt actgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggtaa trpC atgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaa SEQ ID NO: 270 agagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcgacattttt atgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgt caaaaggcgtgatccgtgatgatttcgatccggcacgcattgccgccatttataaacatta cgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttcctccc catcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttacc agatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggat gacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctga ccgaagtcagtaatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgt tggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccgcgagct tgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaatacttac gctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatgg cccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtg gcctgacacgtgggcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgg gttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgcaggaagtgatg gctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcg gacaaagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagct gtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaag tgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaac ggtcagggcgggagcggacaacgtttcgactggtcactattaaatggtcaatcgcttgg caacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcacaaac cggctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgc acgtcttttggcctcggttttccagacgctgcgcgcatattaa trpB atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcct SEQ ID NO: 271 gatgcctgctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatt tcaggctcagttcaacgacctgctgaaaaactatgccgggcgtccaaccgcgctgacca aatgccagaacattacagccgggacgaacaccacgctgtatctgaagcgcgaagattt gctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaag cggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtc ggcccttgccagcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacg ttgaacgccagtcgcccaacgttttccggatgcgcttaatgggtgcggaagtgatcccg gtacatagcggttccgcgaccctgaaagatgcctgtaatgaggcgctacgcgactggtc cggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatccttacc cgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctg gaaagagaaggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgc catcggtatgtttgcagatttcatcaacgaaaccgacgtcggcctgattggtgtggagcct ggcggccacggtatcgaaactggcgagcacggcgcaccgttaaaacatggtcgcgtg ggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaattgaaga gtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctca acagcactggacgcgctgattacgtgtctattaccgacgatgaagccctggaagccttta aaacgctttgcctgcatgaagggatcatcccggcgctggaatcctcccacgccctggcc catgcgctgaaaatgatgcgcgaaaatccggaaaaagagcagctactggtggttaacct ttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagcacgagggga aatctg trpA atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgtt SEQ ID NO: 272 cctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaat tgaagccggtgctgacgcgctggagttaggcatccccttctccgacccactggcggatg gcccgacgattcaaaacgccacactgcgtgcttttgcggcgggagtaaccccggcgca gtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggccttttg atgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaag tcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgc caggccgcgttgcgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatg atttgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcgcgagcggg cgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctg aaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggta aaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaat catcgagcaacatattaatgagccagagaaaatgctggcggcactgaaagcttttgtaca accgatgaaagcggcgacgcgcagttaa fbrS40FTrpE- ctctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccga DCBA (leader ctctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcacc region and RBS agttgtgtggggatcgtccggcaacgctgctgctggaattcgcagatatcgacagcaaa underlined) gatgatttaaaaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgac SEQ ID NO: 273 actgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactggataac gccttgcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcttccc gcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttttgacgcttt ccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcttcgg cggcctgttctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaa aatagctgccctgatttctgatttatctcgctgaaacgctgatggtgattgaccatcagaaa aaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctc actgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccg gtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggt gtagtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctc gccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgctgaaaaagagta atcccagcccgtacatgattttatgcaggataatgatttcaccctgtttggcgcgtcgccg gaaagttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgccgg aacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgc atcgaactggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatc tcgcccgtaatgacctggcacgcatttgcacacccggcagccgctacgtcgccgatctc accaaagttgaccgttactcttacgtgatgcacctagtctcccgcgttgttggtgagctgc gccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagc ggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcg gcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcatt gtgatccgctcggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcg tagtccttgattctgttccgcagtcggaagccgacgaaactcgtaataaagcccgcgctg tactgcgcgctattgccaccgcgcatcatgcacaggagacgttctaatggctgacattct gctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatggt cataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcg acgatgagcaatccggtgctgatgctttctcctggccccggtgtgccgagcgaagccgg ttgtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttgcctc ggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattct tcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaacaa acccgctgccagtggcgcgttatcactcgctggttggcagtaacattccggccggtttaa ccatcaacgcccattttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgttt gtggattccagttccatccggaatccattcttactacccagggcgctcgcctgctggaac aaacgctggcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattc tggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgttttca gcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcat gaaaattcgcggtgaacacccgaacgagatcgccggggcagcaaccgcgctactgga aaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgtcggtactggcgg tgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcg ggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcgga tctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgct ggatgagttaggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatgcg atgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctgggaccattgatt aacccggcgcatccgccgctggcgctaattggtgtttatagtccggaactggtgctgcc gattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgcacagcggc gggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcga aattaagagctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagca attggcaggcggaacaccggaagaaaaccgtgacattttaacacgcttgttacaaggta aaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcct gcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggtactgcgcagtg gttccgcttacgacagagtcaccgcactggcggcacgagggtaaatgatgcaaaccgtt ttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagcagcaac cgctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttca gggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtg atccgtgatgatttcgatccggcacgcattgccgccatttataaacattacgcttcggcaat ttcagtgctgactgatgagaaatattttcaggggagctttgatttcctccccatcgtcagcc aaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctg gcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaat atcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagt aatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgttggcatcaaca accgcgatctgcgcgatttgtcgattgatctcaaccgtacccgcgagcttgcgccgaaac tggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggtgcgc gagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgattt gaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtg ggcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgggttgatttttgttgc gacatcaccgcgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccg ttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcggacaaagctaag gtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaat ctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaact cttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcggg agcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctgg cggggggcttaggcgcagataactgcgtggaagcggcacaaaccggctgcgccggg cttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctc ggttttccagacgctgcgcgcatattaaggaaaggaacaatgacaacattacttaacccc tattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgccagct ggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacct gctgaaaaactatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagcc gggacgaacaccacgctgtatctgaagcgcgaagatttgctgcacggcggcgcgcata aaactaaccaggtgctcggtcaggctttactggcgaagcggatgggtaaaactgaaatt attgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcgccctgc tcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaac gttttccggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgacc ctgaaagatgcctgtaatgaggcgctacgcgactggtccggcagttatgaaaccgcgc actatatgctgggtaccgcagctggcccgcatccttacccgaccattgtgcgtgagtttca gcggatgattggcgaagaaacgaaagcgcagattctggaaagagaaggtcgcctgcc ggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttca tcaacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaac tggcgagcacggcgcaccgttaaaacatggtcgcgtgggcatctatttcggtatgaaag cgccgatgatgcaaaccgaagacgggcaaattgaagagtcttactccatttctgccggg ctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcactggacgcgctgat tacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaag ggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcg cgaaaatccggaaaaagagcagctactggtggttaacctttccggtcgcggcgataaag acatcttcaccgttcacgatattttgaaagcacgaggggaaatctgatggaacgctacga atctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgct gacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattca aaacgccacactgcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgc tggcactcattcgccagaagcacccgaccattcccatcggccttttgatgtatgccaacct ggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcgattc ggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttg cgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgcca gatagcctcttacggtcgtggttacacctatttgctgtcgcgagcgggcgtgaccggcgc agaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacg ctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccgcgattg atgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacata ttaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcg gcgacgcgcagttaa fbrTrpE atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa SEQ ID NO: 274 cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaattc gcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgcg cattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccct gttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactg ccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctc cctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacg agaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaatttac cgcaactgtcagcggaaaatagctgccctgatttctgatttatctcgctgaaacgctgatg gtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaa gaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaag ccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagc gatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaaatt ttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcctatta cgtgctgaaaaagagtaatcccagcccgtacatgattttatgcaggataatgatttcaccc tgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgag atttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacag agacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctgaac atctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagc cgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctccc gcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagca gaaggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatggcg atctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtg caagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaaactcgt aataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagacgtt cta trpDH- ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacga fldABCDacuIfldH tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg (leader region and ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg RBS underlined) aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc SEQ ID NO: 275 ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca gtaaaagcgcgtcgcgggtaaTACTtaagaaggagatatacatATGCTGTT ATTCGAGACTGTGCGTGAAATGGGTCATGAGCAAGT CCTTTTCTGTCATAGCAAGAATCCCGAGATCAAGGCA ATTATCGCAATCCACGATACCACCTTAGGACCGGCTA TGGGCGCAACTCGTATCTTACCTTATATTAATGAGGA GGCTGCCCTGAAAGATGCATTACGTCTGTCCCGCGGA ATGACTTACAAAGCAGCCTGCGCCAATATTCCCGCCG GGGGCGGCAAAGCCGTCATCATCGCTAACCCCGAAA ACAAGACCGATGACCTGTTACGCGCATACGGCCGTTT CGTGGACAGCTTGAACGGCCGTTTCATCACCGGGCAG GACGTTAACATTACGCCCGACGACGTTCGCACTATTT CGCAGGAGACTAAGTACGTGGTAGGCGTCTCAGAAA AGTCGGGAGGGCCGGCACCTATCACCTCTCTGGGAGT ATTTTTAGGCATCAAAGCCGCTGTAGAGTCGCGTTGG CAGTCTAAACGCCTGGATGGCATGAAAGTGGCGGTG CAAGGACTTGGGAACGTAGGAAAAAATCTTTGTCGC CATCTGCATGAACACGATGTACAACTTTTTGTGTCTG ATGTCGATCCAATCAAGGCCGAGGAAGTAAAACGCT TATTCGGGGCGACTGTTGTCGAACCGACTGAAATCTA TTCTTTAGATGTTGATATTTTTGCACCGTGTGCACTTG GGGGTATTTTGAATAGCCATACCATCCCGTTCTTACA AGCCTCAATCATCGCAGGAGCAGCGAATAACCAGCT GGAGAACGAGCAACTTCATTCGCAGATGCTTGCGAA AAAGGGTATTCTTTACTCACCAGACTACGTTATCAAT GCAGGAGGACTTATCAATGTTTATAACGAAATGATCG GATATGACGAGGAAAAAGCATTCAAACAAGTTCATA ACATCTACGATACGTTATTAGCGATTTTCGAAATTGC AAAAGAACAAGGTGTAACCACCAACGACGCGGCCCG TCGTTTAGCAGAGGATCGTATCAACAACTCCAAACGC TCAAAGAGTAAAGCGATTGCGGCGTGAAATGtaagaagg agatatacatATGGAAAACAACACCAATATGTTCTCTGGAG TGAAGGTGATCGAACTGGCCAACTTTATCGCTGCTCC GGCGGCAGGTCGCTTCTTTGCTGATGGGGGAGCAGA AGTAATTAAGATCGAATCTCCAGCAGGCGACCCGCT GCGCTACACGGCCCCATCAGAAGGACGCCCGCTTTCT CAAGAGGAAAACACAACGTATGATTTGGAAAACGCG AATAAGAAAGCAATTGTTCTGAACTTAAAATCGGAA AAAGGAAAGAAAATTCTTCACGAGATGCTTGCTGAG GCAGACATCTTGTTAACAAATTGGCGCACGAAAGCG TTAGTCAAACAGGGGTTAGATTACGAAACACTGAAA GAGAAGTATCCAAAATTGGTATTTGCACAGATTACAG GATACGGGGAGAAAGGACCCGACAAAGACCTGCCTG GTTTCGACTACACGGCGTTTTTCGCCCGCGGAGGAGT CTCCGGTACATTATATGAAAAAGGAACTGTCCCTCCT AATGTGGTACCGGGTCTGGGTGACCACCAGGCAGGA ATGTTCTTAGCTGCCGGTATGGCTGGTGCGTTGTATA AGGCCAAAACCACCGGACAAGGCGACAAAGTCACCG TTAGTCTGATGCATAGCGCAATGTACGGCCTGGGAAT CATGATTCAGGCAGCCCAGTACAAGGACCATGGGCT GGTGTACCCGATCAACCGTAATGAAACGCCTAATCCT TTCATCGTTTCATACAAGTCCAAAGATGATTACTTTG TCCAAGTTTGCATGCCTCCCTATGATGTGTTTTATGAT CGCTTTATGACGGCCTTAGGACGTGAAGACTTGGTAG GTGACGAACGCTACAATAAGATCGAGAACTTGAAGG ATGGTCGCGCAAAAGAAGTCTATTCCATCATCGAACA ACAAATGGTAACGAAGACGAAGGACGAATGGGACA AGATTTTTCGTGATGCAGACATTCCATTCGCTATTGC CCAAACGTGGGAAGATCTTTTAGAAGACGAGCAGGC ATGGGCCAACGACTACCTGTATAAAATGAAGTATCCC ACAGGCAACGAACGTGCCCTGGTACGTTTACCTGTGT TCTTCAAAGAAGCTGGACTTCCTGAATACAACCAGTC GCCACAGATTGCTGAGAATACCGTGGAAGTGTTAAA GGAGATGGGATATACCGAGCAAGAAATTGAGGAGCT TGAGAAAGACAAAGACATCATGGTACGTAAAGAGAA ATGAAGGTtaagaaggagatatacatATGTCAGACCGCAACAA AGAAGTGAAAGAAAAGAAGGCTAAACACTATCTGCG CGAGATCACAGCTAAACACTACAAGGAAGCGTTAGA GGCTAAAGAGCGTGGGGAGAAAGTGGGTTGGTGTGC CTCTAACTTCCCCCAAGAGATTGCAACCACGTTGGGT GTAAAGGTTGTTTATCCCGAAAACCACGCCGCCGCCG TAGCGGCACGTGGCAATGGGCAAAATATGTGCGAAC ACGCGGAGGCTATGGGATTCAGTAATGATGTGTGTG GATATGCACGTGTAAATTTAGCCGTAATGGACATCGG CCATAGTGAAGATCAACCTATTCCAATGCCTGATTTC GTTCTGTGCTGTAATAATATCTGCAATCAGATGATTA AATGGTATGAACACATTGCAAAAACGTTGGATATTCC TATGATCCTTATCGATATTCCATATAATACTGAGAAC ACGGTGTCTCAGGACCGCATTAAGTACATCCGCGCCC AGTTCGATGACGCTATCAAGCAACTGGAAGAAATCA CTGGCAAAAAGTGGGACGAGAATAAATTCGAAGAAG TGATGAAGATTTCGCAAGAATCGGCCAAGCAATGGT TACGCGCCGCGAGCTACGCGAAATACAAACCATCAC CGTTTTCGGGCTTTGACCTTTTTAATCACATGGCTGTA GCCGTTTGTGCTCGCGGCACCCAGGAAGCCGCCGATG CATTCAAAATGTTAGCAGATGAATATGAAGAGAACG TTAAGACAGGAAAGTCTACTTATCGCGGCGAGGAGA AGCAGCGTATCTTGTTCGAGGGCATCGCTTGTTGGCC TTATCTGCGCCACAAGTTGACGAAACTGAGTGAATAT GGAATGAACGTCACAGCTACGGTGTACGCCGAAGCT TTTGGGGTTATTTACGAAAACATGGATGAACTGATGG CCGCTTACAATAAAGTGCCTAACTCAATCTCCTTCGA GAACGCGCTGAAGATGCGTCTTAATGCCGTTACAAGC ACCAATACAGAAGGGGCTGTTATCCACATTAATCGCA GTTGTAAGCTGTGGTCAGGATTCTTATACGAACTGGC CCGTCGTTTGGAAAAGGAGACGGGGATCCCTGTTGTT TCGTTCGACGGAGATCAAGCGGATCCCCGTAACTTCT CCGAGGCTCAATATGACACTCGCATCCAAGGTTTAAA TGAGGTGATGGTCGCGAAAAAAGAAGCAGAGTGAGC TTtaagaaggagatatacatATGTCGAATAGTGACAAGTTTTTT AACGACTTCAAGGACATTGTGGAAAACCCAAAGAAG TATATCATGAAGCATATGGAACAAACGGGACAAAAA GCCATCGGTTGCATGCCTTTATACACCCCAGAAGAGC TTGTCTTAGCGGCGGGTATGTTTCCTGTTGGAGTATG GGGCTCGAATACTGAGTTGTCAAAAGCCAAGACCTA CTTTCCGGCTTTTATCTGTTCTATCTTGCAAACTACTT TAGAAAACGCATTGAATGGGGAGTATGACATGCTGT CTGGTATGATGATCACAAACTATTGCGATTCGCTGAA ATGTATGGGACAAAACTTCAAACTTACAGTGGAAAA TATCGAATTCATCCCGGTTACGGTTCCACAAAACCGC AAGATGGAGGCGGGTAAAGAATTTCTGAAATCCCAG TATAAAATGAATATCGAACAACTGGAAAAAATCTCA GGGAATAAGATCACTGACGAGAGCTTGGAGAAGGCT ATTGAAATTTACGATGAGCACCGTAAAGTCATGAAC GATTTCTCTATGCTTGCGTCCAAGTACCCTGGTATCAT TACGCCAACGAAACGTAACTACGTGATGAAGTCAGC GTATTATATGGACAAGAAAGAACATACAGAGAAGGT ACGTCAGTTGATGGATGAAATCAAGGCCATTGAGCCT AAACCATTCGAAGGAAAACGCGTGATTACCACTGGG ATCATTGCAGATTCGGAGGACCTTTTGAAAATCTTGG AGGAGAATAACATTGCTATCGTGGGAGATGATATTG CACACGAGTCTCGCCAATACCGCACTTTGACCCCGGA GGCCAACACACCTATGGACCGTCTTGCTGAACAATTT GCGAACCGCGAGTGTTCGACGTTGTATGACCCTGAAA AAAAACGTGGACAGTATATTGTCGAGATGGCAAAAG AGCGTAAGGCCGACGGAATCATCTTCTTCATGACAAA ATTCTGCGATCCCGAAGAATACGATTACCCTCAGATG AAAAAAGACTTCGAAGAAGCCGGTATTCCCCACGTT CTGATTGAGACAGACATGCAAATGAAGAACTACGAA CAAGCTCGCACCGCTATTCAAGCATTTTCAGAAACCC TTTGACGCTtaagaaggagatatacatATGCGTGCTGTCTTAAT CGAGAAGTCAGATGACACCCAGAGTGTTTCAGTTAC GGAGTTGGCTGAAGACCAATTACCCGAAGGTGACGT CCTTGTGGATGTCGCGTACAGCACATTGAATTACAAG GATGCTCTTGCGATTACTGGAAAAGCACCCGTTGTAC GCCGTTTTCCTATGGTCCCCGGAATTGACTTTACTGG GACTGTCGCACAGAGTTCCCATGCTGATTTCAAGCCA GGCGACCGCGTAATTCTGAACGGATGGGGAGTTGGT GAGAAACACTGGGGCGGTCTTGCAGAACGCGCACGC GTACGTGGGGACTGGCTTGTCCCGTTGCCAGCCCCCT TAGACTTGCGCCAGGCTGCAATGATTGGCACTGCGGG GTACACAGCTATGCTGTGCGTGCTTGCCCTTGAGCGC CATGGAGTCGTACCTGGGAACGGCGAGATTGTCGTCT CAGGCGCAGCAGGAGGGGTAGGTTCTGTAGCAACCA CACTGTTAGCAGCCAAAGGCTACGAAGTGGCCGCCG TGACCGGGCGCGCAAGCGAGGCCGAATATTTACGCG GATTAGGCGCCGCGTCGGTCATTGATCGCAATGAATT AACGGGGAAGGTGCGTCCATTAGGGCAGGAACGCTG GGCAGGAGGAATCGATGTAGCAGGATCAACCGTACT TGCTAATATGTTGAGCATGATGAAATACCGTGGCGTG GTGGCGGCCTGTGGCCTGGCGGCTGGAATGGACTTGC CCGCGTCTGTCGCCCCTTTTATTCTGCGTGGTATGACT TTGGCAGGGGTAGATTCAGTCATGTGCCCCAAAACTG ATCGTCTGGCTGCTTGGGCACGCCTGGCATCCGACCT GGACCCTGCAAAGCTGGAAGAGATGACAACTGAATT ACCGTTCTCTGAGGTGATTGAAACGGCTCCGAAGTTC TTGGATGGAACAGTGCGTGGGCGTATTGTCATTCCGG TAACACCTTGATACTtaagaaggagatatacatATGAAAATCTT GGCATACTGCGTCCGCCCAGACGAGGTAGACTCCTTT AAGAAATTTAGTGAAAAGTACGGGCATACAGTTGAT CTTATTCCAGACTCTTTTGGACCTAATGTCGCTCATTT GGCGAAGGGTTACGATGGGATTTCTATTCTGGGCAAC GACACGTGTAACCGTGAGGCACTGGAGAAGATCAAG GATTGCGGGATCAAATATCTGGCAACCCGTACAGCC GGAGTGAACAACATTGACTTCGATGCAGCAAAGGAG TTCGGTATTAACGTGGCTAATGTTCCCGCATATTCCC CCAACTCGGTCAGCGAATTTACCATTGGATTGGCATT AAGTCTGACGCGTAAGATTCCATTTGCCCTGAAACGC GTGGAACTGAACAATTTTGCGCTTGGCGGCCTTATTG GTGTGGAATTGCGTAACTTAACTTTAGGAGTCATCGG TACTGGTCGCATCGGATTGAAAGTGATTGAGGGCTTC TCTGGGTTTGGAATGAAAAAAATGATCGGTTATGACA TTTTTGAAAATGAAGAAGCAAAGAAGTACATCGAAT ACAAATCATTAGACGAAGTTTTTAAAGAGGCTGATAT TATCACTCTGCATGCGCCTCTGACAGACGACAACTAT CATATGATTGGTAAAGAATCCATTGCTAAAATGAAG GATGGGGTATTTATTATCAACGCAGCGCGTGGAGCCT TAATCGATAGTGAGGCCCTGATTGAAGGGTTAAAATC GGGGAAGATT fldA ATGGAAAACAACACCAATATGTTCTCTGGAGTGAAG SEQ ID NO: 276 GTGATCGAACTGGCCAACTTTATCGCTGCTCCGGCGG CAGGTCGCTTCTTTGCTGATGGGGGAGCAGAAGTAAT TAAGATCGAATCTCCAGCAGGCGACCCGCTGCGCTAC ACGGCCCCATCAGAAGGACGCCCGCTTTCTCAAGAG GAAAACACAACGTATGATTTGGAAAACGCGAATAAG AAAGCAATTGTTCTGAACTTAAAATCGGAAAAAGGA AAGAAAATTCTTCACGAGATGCTTGCTGAGGCAGAC ATCTTGTTAACAAATTGGCGCACGAAAGCGTTAGTCA AACAGGGGTTAGATTACGAAACACTGAAAGAGAAGT ATCCAAAATTGGTATTTGCACAGATTACAGGATACGG GGAGAAAGGACCCGACAAAGACCTGCCTGGTTTCGA CTACACGGCGTTTTTCGCCCGCGGAGGAGTCTCCGGT ACATTATATGAAAAAGGAACTGTCCCTCCTAATGTGG TACCGGGTCTGGGTGACCACCAGGCAGGAATGTTCTT AGCTGCCGGTATGGCTGGTGCGTTGTATAAGGCCAAA ACCACCGGACAAGGCGACAAAGTCACCGTTAGTCTG ATGCATAGCGCAATGTACGGCCTGGGAATCATGATTC AGGCAGCCCAGTACAAGGACCATGGGCTGGTGTACC CGATCAACCGTAATGAAACGCCTAATCCTTTCATCGT TTCATACAAGTCCAAAGATGATTACTTTGTCCAAGTT TGCATGCCTCCCTATGATGTGTTTTATGATCGCTTTAT GACGGCCTTAGGACGTGAAGACTTGGTAGGTGACGA ACGCTACAATAAGATCGAGAACTTGAAGGATGGTCG CGCAAAAGAAGTCTATTCCATCATCGAACAACAAAT GGTAACGAAGACGAAGGACGAATGGGACAAGATTTT TCGTGATGCAGACATTCCATTCGCTATTGCCCAAACG TGGGAAGATCTTTTAGAAGACGAGCAGGCATGGGCC AACGACTACCTGTATAAAATGAAGTATCCCACAGGC AACGAACGTGCCCTGGTACGTTTACCTGTGTTCTTCA AAGAAGCTGGACTTCCTGAATACAACCAGTCGCCAC AGATTGCTGAGAATACCGTGGAAGTGTTAAAGGAGA TGGGATATACCGAGCAAGAAATTGAGGAGCTTGAGA AAGACAAAGACATCATGGTACGTAAAGAGAAATGA fldB ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAA SEQ ID NO: 277 GGCTAAACACTATCTGCGCGAGATCACAGCTAAACA CTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGGGGA GAAAGTGGGTTGGTGTGCCTCTAACTTCCCCCAAGAG ATTGCAACCACGTTGGGTGTAAAGGTTGTTTATCCCG AAAACCACGCCGCCGCCGTAGCGGCACGTGGCAATG GGCAAAATATGTGCGAACACGCGGAGGCTATGGGAT TCAGTAATGATGTGTGTGGATATGCACGTGTAAATTT AGCCGTAATGGACATCGGCCATAGTGAAGATCAACC TATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAATA TCTGCAATCAGATGATTAAATGGTATGAACACATTGC AAAAACGTTGGATATTCCTATGATCCTTATCGATATT CCATATAATACTGAGAACACGGTGTCTCAGGACCGC ATTAAGTACATCCGCGCCCAGTTCGATGACGCTATCA AGCAACTGGAAGAAATCACTGGCAAAAAGTGGGACG AGAATAAATTCGAAGAAGTGATGAAGATTTCGCAAG AATCGGCCAAGCAATGGTTACGCGCCGCGAGCTACG CGAAATACAAACCATCACCGTTTTCGGGCTTTGACCT TTTTAATCACATGGCTGTAGCCGTTTGTGCTCGCGGC ACCCAGGAAGCCGCCGATGCATTCAAAATGTTAGCA GATGAATATGAAGAGAACGTTAAGACAGGAAAGTCT ACTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCG AGGGCATCGCTTGTTGGCCTTATCTGCGCCACAAGTT GACGAAACTGAGTGAATATGGAATGAACGTCACAGC TACGGTGTACGCCGAAGCTTTTGGGGTTATTTACGAA AACATGGATGAACTGATGGCCGCTTACAATAAAGTG CCTAACTCAATCTCCTTCGAGAACGCGCTGAAGATGC GTCTTAATGCCGTTACAAGCACCAATACAGAAGGGG CTGTTATCCACATTAATCGCAGTTGTAAGCTGTGGTC AGGATTCTTATACGAACTGGCCCGTCGTTTGGAAAAG GAGACGGGGATCCCTGTTGTTTCGTTCGACGGAGATC AAGCGGATCCCCGTAACTTCTCCGAGGCTCAATATGA CACTCGCATCCAAGGTTTAAATGAGGTGATGGTCGCG AAAAAAGAAGCAGAGTGA fldC ATGTCGAATAGTGACAAGTTTTTTAACGACTTCAAGG SEQ ID NO: 278 ACATTGTGGAAAACCCAAAGAAGTATATCATGAAGC ATATGGAACAAACGGGACAAAAAGCCATCGGTTGCA TGCCTTTATACACCCCAGAAGAGCTTGTCTTAGCGGC GGGTATGTTTCCTGTTGGAGTATGGGGCTCGAATACT GAGTTGTCAAAAGCCAAGACCTACTTTCCGGCTTTTA TCTGTTCTATCTTGCAAACTACTTTAGAAAACGCATT GAATGGGGAGTATGACATGCTGTCTGGTATGATGATC ACAAACTATTGCGATTCGCTGAAATGTATGGGACAA AACTTCAAACTTACAGTGGAAAATATCGAATTCATCC CGGTTACGGTTCCACAAAACCGCAAGATGGAGGCGG GTAAAGAATTTCTGAAATCCCAGTATAAAATGAATAT CGAACAACTGGAAAAAATCTCAGGGAATAAGATCAC TGACGAGAGCTTGGAGAAGGCTATTGAAATTTACGA TGAGCACCGTAAAGTCATGAACGATTTCTCTATGCTT GCGTCCAAGTACCCTGGTATCATTACGCCAACGAAAC GTAACTACGTGATGAAGTCAGCGTATTATATGGACAA GAAAGAACATACAGAGAAGGTACGTCAGTTGATGGA TGAAATCAAGGCCATTGAGCCTAAACCATTCGAAGG AAAACGCGTGATTACCACTGGGATCATTGCAGATTCG GAGGACCTTTTGAAAATCTTGGAGGAGAATAACATT GCTATCGTGGGAGATGATATTGCACACGAGTCTCGCC AATACCGCACTTTGACCCCGGAGGCCAACACACCTAT GGACCGTCTTGCTGAACAATTTGCGAACCGCGAGTGT TCGACGTTGTATGACCCTGAAAAAAAACGTGGACAG TATATTGTCGAGATGGCAAAAGAGCGTAAGGCCGAC GGAATCATCTTCTTCATGACAAAATTCTGCGATCCCG AAGAATACGATTACCCTCAGATGAAAAAAGACTTCG AAGAAGCCGGTATTCCCCACGTTCTGATTGAGACAGA CATGCAAATGAAGAACTACGAACAAGCTCGCACCGC TATTCAAGCATTTTCAGAAACCCTTTG AcuI ATGCGTGCTGTCTTAATCGAGAAGTCAGATGACACCC SEQ ID NO: 279 AGAGTGTTTCAGTTACGGAGTTGGCTGAAGACCAATT ACCCGAAGGTGACGTCCTTGTGGATGTCGCGTACAGC ACATTGAATTACAAGGATGCTCTTGCGATTACTGGAA AAGCACCCGTTGTACGCCGTTTTCCTATGGTCCCCGG AATTGACTTTACTGGGACTGTCGCACAGAGTTCCCAT GCTGATTTCAAGCCAGGCGACCGCGTAATTCTGAACG GATGGGGAGTTGGTGAGAAACACTGGGGCGGTCTTG CAGAACGCGCACGCGTACGTGGGGACTGGCTTGTCC CGTTGCCAGCCCCCTTAGACTTGCGCCAGGCTGCAAT GATTGGCACTGCGGGGTACACAGCTATGCTGTGCGTG CTTGCCCTTGAGCGCCATGGAGTCGTACCTGGGAACG GCGAGATTGTCGTCTCAGGCGCAGCAGGAGGGGTAG GTTCTGTAGCAACCACACTGTTAGCAGCCAAAGGCTA CGAAGTGGCCGCCGTGACCGGGCGCGCAAGCGAGGC CGAATATTTACGCGGATTAGGCGCCGCGTCGGTCATT GATCGCAATGAATTAACGGGGAAGGTGCGTCCATTA GGGCAGGAACGCTGGGCAGGAGGAATCGATGTAGCA GGATCAACCGTACTTGCTAATATGTTGAGCATGATGA AATACCGTGGCGTGGTGGCGGCCTGTGGCCTGGCGG CTGGAATGGACTTGCCCGCGTCTGTCGCCCCTTTTATT CTGCGTGGTATGACTTTGGCAGGGGTAGATTCAGTCA TGTGCCCCAAAACTGATCGTCTGGCTGCTTGGGCACG CCTGGCATCCGACCTGGACCCTGCAAAGCTGGAAGA GATGACAACTGAATTACCGTTCTCTGAGGTGATTGAA ACGGCTCCGAAGTTCTTGGATGGAACAGTGCGTGGG CGTATTGTCATTCCGGTAACACCTTGA fldH1 ATGAAAATCTTGGCATACTGCGTCCGCCCAGACGAG SEQ ID NO: 280 GTAGACTCCTTTAAGAAATTTAGTGAAAAGTACGGGC ATACAGTTGATCTTATTCCAGACTCTTTTGGACCTAAT GTCGCTCATTTGGCGAAGGGTTACGATGGGATTTCTA TTCTGGGCAACGACACGTGTAACCGTGAGGCACTGG AGAAGATCAAGGATTGCGGGATCAAATATCTGGCAA CCCGTACAGCCGGAGTGAACAACATTGACTTCGATGC AGCAAAGGAGTTCGGTATTAACGTGGCTAATGTTCCC GCATATTCCCCCAACTCGGTCAGCGAATTTACCATTG GATTGGCATTAAGTCTGACGCGTAAGATTCCATTTGC CCTGAAACGCGTGGAACTGAACAATTTTGCGCTTGGC GGCCTTATTGGTGTGGAATTGCGTAACTTAACTTTAG GAGTCATCGGTACTGGTCGCATCGGATTGAAAGTGAT TGAGGGCTTCTCTGGGTTTGGAATGAAAAAAATGATC GGTTATGACATTTTTGAAAATGAAGAAGCAAAGAAG TACATCGAATACAAATCATTAGACGAAGTTTTTAAAG AGGCTGATATTATCACTCTGCATGCGCCTCTGACAGA CGACAACTATCATATGATTGGTAAAGAATCCATTGCT AAAATGAAGGATGGGGTATTTATTATCAACGCAGCG CGTGGAGCCTTAATCGATAGTGAGGCCCTGATTGAAG GGTTAAAATCGGGGAAGATTGCGGGCGCGGCTCTGG ATAGCTATGAGTATGAGCAAGGTGTCTTTCACAACAA TAAGATGAATGAAATTATGCAGGATGATACCTTGGA ACGTCTGAAATCTTTTCCCAACGTCGTGATCACGCCG CATTTGGGTTTTTATACTGATGAGGCGGTTTCCAATA TGGTAGAGATCACACTGATGAACCTTCAGGAATTCGA GTTGAAAGGAACCTGTAAGAACCAGCGTGTTTGTAA ATGA fbrAroG-TrpDH- Ctctagaaataattttgtttaactttaagaaggagatatacat fldABCDH (RBS atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg and leader region cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga underlined) aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca SEQ ID NO: 281 tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACTtaagaaggaga tatacatATGCTGTTATTCGAGACTGTGCGTGAAATGGGT CATGAGCAAGTCCTTTTCTGTCATAGCAAGAATCCCG AGATCAAGGCAATTATCGCAATCCACGATACCACCTT AGGACCGGCTATGGGCGCAACTCGTATCTTACCTTAT ATTAATGAGGAGGCTGCCCTGAAAGATGCATTACGTC TGTCCCGCGGAATGACTTACAAAGCAGCCTGCGCCA ATATTCCCGCCGGGGGCGGCAAAGCCGTCATCATCGC TAACCCCGAAAACAAGACCGATGACCTGTTACGCGC ATACGGCCGTTTCGTGGACAGCTTGAACGGCCGTTTC ATCACCGGGCAGGACGTTAACATTACGCCCGACGAC GTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAG GCGTCTCAGAAAAGTCGGGAGGGCCGGCACCTATCA CCTCTCTGGGAGTATTTTTAGGCATCAAAGCCGCTGT AGAGTCGCGTTGGCAGTCTAAACGCCTGGATGGCAT GAAAGTGGCGGTGCAAGGACTTGGGAACGTAGGAAA AAATCTTTGTCGCCATCTGCATGAACACGATGTACAA CTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGAGG AAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACC GACTGAAATCTATTCTTTAGATGTTGATATTTTTGCAC CGTGTGCACTTGGGGGTATTTTGAATAGCCATACCAT CCCGTTCTTACAAGCCTCAATCATCGCAGGAGCAGCG AATAACCAGCTGGAGAACGAGCAACTTCATTCGCAG ATGCTTGCGAAAAAGGGTATTCTTTACTCACCAGACT ACGTTATCAATGCAGGAGGACTTATCAATGTTTATAA CGAAATGATCGGATATGACGAGGAAAAAGCATTCAA ACAAGTTCATAACATCTACGATACGTTATTAGCGATT TTCGAAATTGCAAAAGAACAAGGTGTAACCACCAAC GACGCGGCCCGTCGTTTAGCAGAGGATCGTATCAAC AACTCCAAACGCTCAAAGAGTAAAGCGATTGCGGCG TGAAATGtaagaaggagatatacatATGGAAAACAACACCAAT ATGTTCTCTGGAGTGAAGGTGATCGAACTGGCCAACT TTATCGCTGCTCCGGCGGCAGGTCGCTTCTTTGCTGA TGGGGGAGCAGAAGTAATTAAGATCGAATCTCCAGC AGGCGACCCGCTGCGCTACACGGCCCCATCAGAAGG ACGCCCGCTTTCTCAAGAGGAAAACACAACGTATGA TTTGGAAAACGCGAATAAGAAAGCAATTGTTCTGAA CTTAAAATCGGAAAAAGGAAAGAAAATTCTTCACGA GATGCTTGCTGAGGCAGACATCTTGTTAACAAATTGG CGCACGAAAGCGTTAGTCAAACAGGGGTTAGATTAC GAAACACTGAAAGAGAAGTATCCAAAATTGGTATTT GCACAGATTACAGGATACGGGGAGAAAGGACCCGAC AAAGACCTGCCTGGTTTCGACTACACGGCGTTTTTCG CCCGCGGAGGAGTCTCCGGTACATTATATGAAAAAG GAACTGTCCCTCCTAATGTGGTACCGGGTCTGGGTGA CCACCAGGCAGGAATGTTCTTAGCTGCCGGTATGGCT GGTGCGTTGTATAAGGCCAAAACCACCGGACAAGGC GACAAAGTCACCGTTAGTCTGATGCATAGCGCAATGT ACGGCCTGGGAATCATGATTCAGGCAGCCCAGTACA AGGACCATGGGCTGGTGTACCCGATCAACCGTAATG AAACGCCTAATCCTTTCATCGTTTCATACAAGTCCAA AGATGATTACTTTGTCCAAGTTTGCATGCCTCCCTAT GATGTGTTTTATGATCGCTTTATGACGGCCTTAGGAC GTGAAGACTTGGTAGGTGACGAACGCTACAATAAGA TCGAGAACTTGAAGGATGGTCGCGCAAAAGAAGTCT ATTCCATCATCGAACAACAAATGGTAACGAAGACGA AGGACGAATGGGACAAGATTTTTCGTGATGCAGACA TTCCATTCGCTATTGCCCAAACGTGGGAAGATCTTTT AGAAGACGAGCAGGCATGGGCCAACGACTACCTGTA TAAAATGAAGTATCCCACAGGCAACGAACGTGCCCT GGTACGTTTACCTGTGTTCTTCAAAGAAGCTGGACTT CCTGAATACAACCAGTCGCCACAGATTGCTGAGAAT ACCGTGGAAGTGTTAAAGGAGATGGGATATACCGAG CAAGAAATTGAGGAGCTTGAGAAAGACAAAGACATC ATGGTACGTAAAGAGAAATGAAGGTtaagaaggagatatacat ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAA GGCTAAACACTATCTGCGCGAGATCACAGCTAAACA CTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGGGGA GAAAGTGGGTTGGTGTGCCTCTAACTTCCCCCAAGAG ATTGCAACCACGTTGGGTGTAAAGGTTGTTTATCCCG AAAACCACGCCGCCGCCGTAGCGGCACGTGGCAATG GGCAAAATATGTGCGAACACGCGGAGGCTATGGGAT TCAGTAATGATGTGTGTGGATATGCACGTGTAAATTT AGCCGTAATGGACATCGGCCATAGTGAAGATCAACC TATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAATA TCTGCAATCAGATGATTAAATGGTATGAACACATTGC AAAAACGTTGGATATTCCTATGATCCTTATCGATATT CCATATAATACTGAGAACACGGTGTCTCAGGACCGC ATTAAGTACATCCGCGCCCAGTTCGATGACGCTATCA AGCAACTGGAAGAAATCACTGGCAAAAAGTGGGACG AGAATAAATTCGAAGAAGTGATGAAGATTTCGCAAG AATCGGCCAAGCAATGGTTACGCGCCGCGAGCTACG CGAAATACAAACCATCACCGTTTTCGGGCTTTGACCT TTTTAATCACATGGCTGTAGCCGTTTGTGCTCGCGGC ACCCAGGAAGCCGCCGATGCATTCAAAATGTTAGCA GATGAATATGAAGAGAACGTTAAGACAGGAAAGTCT ACTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCG AGGGCATCGCTTGTTGGCCTTATCTGCGCCACAAGTT GACGAAACTGAGTGAATATGGAATGAACGTCACAGC TACGGTGTACGCCGAAGCTTTTGGGGTTATTTACGAA AACATGGATGAACTGATGGCCGCTTACAATAAAGTG CCTAACTCAATCTCCTTCGAGAACGCGCTGAAGATGC GTCTTAATGCCGTTACAAGCACCAATACAGAAGGGG CTGTTATCCACATTAATCGCAGTTGTAAGCTGTGGTC AGGATTCTTATACGAACTGGCCCGTCGTTTGGAAAAG GAGACGGGGATCCCTGTTGTTTCGTTCGACGGAGATC AAGCGGATCCCCGTAACTTCTCCGAGGCTCAATATGA CACTCGCATCCAAGGTTTAAATGAGGTGATGGTCGCG AAAAAAGAAGCAGAGTGAGCTTtaagaaggagatatacatATG TCGAATAGTGACAAGTTTTTTAACGACTTCAAGGACA TTGTGGAAAACCCAAAGAAGTATATCATGAAGCATA TGGAACAAACGGGACAAAAAGCCATCGGTTGCATGC CTTTATACACCCCAGAAGAGCTTGTCTTAGCGGCGGG TATGTTTCCTGTTGGAGTATGGGGCTCGAATACTGAG TTGTCAAAAGCCAAGACCTACTTTCCGGCTTTTATCT GTTCTATCTTGCAAACTACTTTAGAAAACGCATTGAA TGGGGAGTATGACATGCTGTCTGGTATGATGATCACA AACTATTGCGATTCGCTGAAATGTATGGGACAAAACT TCAAACTTACAGTGGAAAATATCGAATTCATCCCGGT TACGGTTCCACAAAACCGCAAGATGGAGGCGGGTAA AGAATTTCTGAAATCCCAGTATAAAATGAATATCGAA CAACTGGAAAAAATCTCAGGGAATAAGATCACTGAC GAGAGCTTGGAGAAGGCTATTGAAATTTACGATGAG CACCGTAAAGTCATGAACGATTTCTCTATGCTTGCGT CCAAGTACCCTGGTATCATTACGCCAACGAAACGTAA CTACGTGATGAAGTCAGCGTATTATATGGACAAGAA AGAACATACAGAGAAGGTACGTCAGTTGATGGATGA AATCAAGGCCATTGAGCCTAAACCATTCGAAGGAAA ACGCGTGATTACCACTGGGATCATTGCAGATTCGGAG GACCTTTTGAAAATCTTGGAGGAGAATAACATTGCTA TCGTGGGAGATGATATTGCACACGAGTCTCGCCAATA CCGCACTTTGACCCCGGAGGCCAACACACCTATGGAC CGTCTTGCTGAACAATTTGCGAACCGCGAGTGTTCGA CGTTGTATGACCCTGAAAAAAAACGTGGACAGTATA TTGTCGAGATGGCAAAAGAGCGTAAGGCCGACGGAA TCATCTTCTTCATGACAAAATTCTGCGATCCCGAAGA ATACGATTACCCTCAGATGAAAAAAGACTTCGAAGA AGCCGGTATTCCCCACGTTCTGATTGAGACAGACATG CAAATGAAGAACTACGAACAAGCTCGCACCGCTATT CAAGCATTTTCAGAAACCCTTTGACGCTtaagaaggagatata catATGTTCTTTACGGAGCAACACGAACTTATTCGCAA ACTGGCGCGTGACTTTGCCGAACAGGAAATCGAGCC TATCGCAGACGAAGTAGATAAAACCGCAGAGTTCCC AAAAGAAATCGTGAAGAAGATGGCTCAAAATGGATT TTTCGGCATTAAAATGCCTAAAGAATACGGAGGGGC GGGTGCGGATAACCGCGCTTATGTCACTATTATGGAG GAAATTTCACGTGCTTCCGGGGTAGCGGGTATCTACC TGAGCTCGCCGAACAGTTTGTTAGGAACTCCCTTCTT ATTGGTCGGAACCGATGAGCAAAAAGAAAAGTACCT TAAGCCTATGATCCGCGGCGAGAAGACTCTGGCGTTC GCCCTGACAGAGCCTGGTGCTGGCTCTGATGCGGGTG CGTTGGCTACTACTGCCCGTGAAGAGGGCGACTATTA TATCTTAAATGGCCGCAAGACGTTTATTACAGGGGCT CCTATTAGCGACAATATTATTGTGTTCGCAAAAACCG ATATGAGCAAAGGGACCAAAGGTATCACCACTTTCA TTGTGGACTCAAAGCAGGAAGGGGTAAGTTTTGGTA AGCCAGAGGACAAAATGGGAATGATTGGTTGTCCGA CAAGCGACATCATCTTGGAAAACGTTAAAGTTCATAA GTCCGACATCTTGGGAGAAGTCAATAAGGGGTTTATT ACCGCGATGAAAACACTTTCCGTTGGTCGTATCGGAG TGGCGTCACAGGCGCTTGGAATTGCACAGGCCGCCGT AGATGAGGCGGTAAAGTACGCCAAGCAACGTAAACA ATTCAATCGCCCAATCGCGAAATTTCAGGCCATTCAA TTTAAACTTGCCAATATGGAGACTAAATTAAATGCCG CTAAACTTCTTGTTTATAACGCAGCGTACAAAATGGA TTGTGGAGAAAAAGCCGACAAGGAAGCCTCTATGGC TAAATACTTTGCTGCTGAATCAGCGATCCAAATCGTT AACGACGCGCTGCAAATCCATGGCGGGTATGGCTAT ATCAAAGACTACAAGATTGAACGTTTGTACCGCGATG TGCGTGTGATCGCTATTTATGAGGGCACTTCCGAGGT CCAACAGATGGTTATCGCGTCCAATCTGCTGAAGTAA TACTtaagaaggagatatacatATGAAAATCTTGGCATACTGCG TCCGCCCAGACGAGGTAGACTCCTTTAAGAAATTTAG TGAAAAGTACGGGCATACAGTTGATCTTATTCCAGAC TCTTTTGGACCTAATGTCGCTCATTTGGCGAAGGGTT ACGATGGGATTTCTATTCTGGGCAACGACACGTGTAA CCGTGAGGCACTGGAGAAGATCAAGGATTGCGGGAT CAAATATCTGGCAACCCGTACAGCCGGAGTGAACAA CATTGACTTCGATGCAGCAAAGGAGTTCGGTATTAAC GTGGCTAATGTTCCCGCATATTCCCCCAACTCGGTCA GCGAATTTACCATTGGATTGGCATTAAGTCTGACGCG TAAGATTCCATTTGCCCTGAAACGCGTGGAACTGAAC AATTTTGCGCTTGGCGGCCTTATTGGTGTGGAATTGC GTAACTTAACTTTAGGAGTCATCGGTACTGGTCGCAT CGGATTGAAAGTGATTGAGGGCTTCTCTGGGTTTGGA ATGAAAAAAATGATCGGTTATGACATTTTTGAAAATG AAGAAGCAAAGAAGTACATCGAATACAAATCATTAG ACGAAGTTTTTAAAGAGGCTGATATTATCACTCTGCA TGCGCCTCTGACAGACGACAACTATCATATGATTGGT AAAGAATCCATTGCTAAAATGAAGGATGGGGTATTT ATTATCAACGCAGCGCGTGGAGCCTTAATCGATAGTG AGGCCCTGATTGAAGGGTTAAAATCGGGGAAGATTG CGGGCGCGGCTCTGGATAGCTATGAGTATGAGCAAG GTGTCTTTCACAACAATAAGATGAATGAAATTATGCA GGATGATACCTTGGAACGTCTGAAATCTTTTCCCAAC GTCGTGATCACGCCGCATTTGGGTTTTTATACTGATG AGGCGGTTTCCAATATGGTAGAGATCACACTGATGA ACCTTCAGGAATTCGAGTTGAAAGGAACCTGTAAGA ACCAGCGTGTTTGTAAATGA FldD ATGTTCTTTACGGAGCAACACGAACTTATTCGCAAAC SEQ ID NO: 282 TGGCGCGTGACTTTGCCGAACAGGAAATCGAGCCTAT CGCAGACGAAGTAGATAAAACCGCAGAGTTCCCAAA AGAAATCGTGAAGAAGATGGCTCAAAATGGATTTTT CGGCATTAAAATGCCTAAAGAATACGGAGGGGCGGG TGCGGATAACCGCGCTTATGTCACTATTATGGAGGAA ATTTCACGTGCTTCCGGGGTAGCGGGTATCTACCTGA GCTCGCCGAACAGTTTGTTAGGAACTCCCTTCTTATT GGTCGGAACCGATGAGCAAAAAGAAAAGTACCTTAA GCCTATGATCCGCGGCGAGAAGACTCTGGCGTTCGCC CTGACAGAGCCTGGTGCTGGCTCTGATGCGGGTGCGT TGGCTACTACTGCCCGTGAAGAGGGCGACTATTATAT CTTAAATGGCCGCAAGACGTTTATTACAGGGGCTCCT ATTAGCGACAATATTATTGTGTTCGCAAAAACCGATA TGAGCAAAGGGACCAAAGGTATCACCACTTTCATTGT GGACTCAAAGCAGGAAGGGGTAAGTTTTGGTAAGCC AGAGGACAAAATGGGAATGATTGGTTGTCCGACAAG CGACATCATCTTGGAAAACGTTAAAGTTCATAAGTCC GACATCTTGGGAGAAGTCAATAAGGGGTTTATTACCG CGATGAAAACACTTTCCGTTGGTCGTATCGGAGTGGC GTCACAGGCGCTTGGAATTGCACAGGCCGCCGTAGA TGAGGCGGTAAAGTACGCCAAGCAACGTAAACAATT CAATCGCCCAATCGCGAAATTTCAGGCCATTCAATTT AAACTTGCCAATATGGAGACTAAATTAAATGCCGCTA AACTTCTTGTTTATAACGCAGCGTACAAAATGGATTG TGGAGAAAAAGCCGACAAGGAAGCCTCTATGGCTAA ATACTTTGCTGCTGAATCAGCGATCCAAATCGTTAAC GACGCGCTGCAAATCCATGGCGGGTATGGCTATATCA AAGACTACAAGATTGAACGTTTGTACCGCGATGTGCG TGTGATCGCTATTTATGAGGGCACTTCCGAGGTCCAA CAGATGGTTATCGCGTCCAATCTGCTGAAGTAA RBS taagaaggagatatacat SEQ ID NO: 283 RBS ctctagaaataattttgtttaactttaagaaggagatatacat SEQ ID NO: 284

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 59. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 59. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 59. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 59. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 59. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences of Table 59. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 263. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 59.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 263. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 263. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 263. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 263. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 263. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 263. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 263. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 263.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 261. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 261. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 261. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 261. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 261. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 261. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 261. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 261.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 273. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 273. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 273. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 273. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 273. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 273.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 256. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 256. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 256.

In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 257. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 257. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 257. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 257. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 257. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 257. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 257. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 257.

Example 38 Tryptophan Production in an Engineered Strain of E. coli Nissle

A number of tryptophan metabolites, either host-derived (such as tryptamine or kynurenine) or intestinal bacteria-derived (such as indole acetate or indole), have been shown to downregulate inflammation and promote gut barrier health, via the activation of the AhR receptor. Other tryptophan metabolites, such as the bacteria-derived indole propionate, have been shown to help restore intestinal barrier integrity, in experimental models of colitis. In this example, the E. coli strain Nissle was engineered to produce tryptophan, the precursor to all those beneficial metabolites.

First, in order to remove the negative regulation of tryptophan biosynthetic genes mediated by the transcription factor TrpR, the trpR gene was deleted form the E. coli Nissle genome. The tryptophan operon trpEDCBA was amplified by PCR from the E. coli Nissle genomic DNA and cloned in the low-copy plasmid pSC101 under the control of the tet promoter, downstream of the tetR repressor gene. This tet-trpEDCBA plasmid was then transformed into the ΔtrpR mutant to obtain the ΔtrpR, tet-trpEDCBA strain. Subsequently, a feedback resistant version of the aroG gene (aroG^(fbr)) from E. coli Nissle, coding for the enzyme catalyzing the first committing step towards aromatic amino acid production, was synthetized and cloned into the medium copy plasmid p15A, under the control of the tet promoter, downstream of the tetR repressor. This plasmid was transformed into the ΔtrpR, tet-trpEDCBA strain to obtain the ΔtrpR, tet-trpEDCBA, tet-aroG^(fbr) strain. Finally, a feedback resistant version of the tet-trpEBCDA construct (tet-trpE^(fbr)BCDA) was generated from the tet-trpEBCDA. Both the tet-aroG^(fbr) and the tet-trpE^(fbr)BCDA constructs were transformed into the ΔtrpR mutant to obtain the ΔtrpR, tet-trpE^(fbr)DCBA, tet-aroG^(fbr) strain.

All generated strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 5 min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 0.5% glucose and placed at 37 C at 250 rpm. 200 uL were collected at 2 h, 4 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 41A shows that tryptophan is being produced and secreted by the ΔtrpR, tet-trpEDCBA, tet-aroG^(fbr) strain. The production of tryptophan is significantly enhanced by expressing the feedback resistant version of trpE.

Example 39 Improved Tryptophan by Using a non-PTS Carbon Source and by Deleting the tnaA Gene Encoding Tryptophanase

One of the precursor molecule to tryptophan in E. coli is phosphoenolpyruvate (PEP). Only 3% of available PEP is normally used to produce aromatic acids (that include tryptophan, phenylalanine and tyrosine). When E. coli is grown using glucose as a sole carbon source, 50% of PEP is used to import glucose into the cell using the phosphotransferase system (PTS). In order to increase tryptophan production, a non-PTS oxidized sugar, glucuronate, was used to test tryptophan secretion by the engineered E. coli Nissle strain ΔrpR, tet-trpe^(fbr)DCBA, tet-aroG^(fbr). In addition, the tnaA gene, encoding the tryptophanase enzyme, was deleted in the ΔtrpR, tet-trpE^(fbr)DCBA, tet-aroG^(fbr) strain in order to block the conversion of tryptophan into indole to obtain the ΔtrpRΔtnaA, tet-trpE ^(fbr)DCBA, tet-aroG^(fbr) strain.

the ΔtrpR, tet-trpE^(fbr)DCBA, tet-aroG^(fbr) and ΔtrpRΔtnaA, tet-trpE^(fbr)DCBA, tet-arod^(br) strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 1% glucose or 1% glucuronate and placed at 37 C at 250 rpm or at 37 C in an anaerobic chamber. 200 uL were collected at 3 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 41B shows that tryptophan production is doubled in aerobic condition when the non-PTS oxidized sugar glucoronate was used. In addition, the deletion of tnaA had a positive effect on tryptophan production at the 3 h time point in both aerobic and anaerobic conditions and at the 16 h time point, only in anaerobic condition.

Example 40 Improved Tryptophan Production by Increasing the Rate of Serine Biosynthesis in E. coli Nissle

The last step in the tryptophan biosynthesis in E. coli consumes one molecule of serine. In this example, we demonstrate that serine availability is a limiting factor for tryptophan production and describe the construction of the tryptophan producing E. coli Nissle strains ΔtrpRΔtnaA, tet-trpE^(fbr)DCBA, tet-aroG^(fbr)serA and ΔtrpRΔtnaA, tet-trpE^(fbr)DCBA, tet-aroG^(fbr)serA^(fbr) strains.

the ΔtrpRΔtnaA, tet-trpE^(fbr)DCBA, tet-aroG^(fbr) strain was grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 5 min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 1% glucuronate or 1% glucuronate and 10 mM serine and placed at 37 C an anaerobic chamber. 200 uL were collected at 3 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 41C shows that tryptophan production is improved three-fold by serine addition.

In order to increase the rate of serine biosynthesis in the ΔtrpRΔtnaA, tet-trpE ^(fbr)DCBA, tet-aroG^(fbr) strain, the serA gene from E. coli Nissle encoding the enzyme catalyzing the first step in the serine biosynthetic pathway was amplified by PCR and cloned into the tet-aroG^(fbr) plasmid by Gibson assembly. The newly generated tet-aroG^(fbr)-serA construct was then transformed into a ΔtrpRΔtnaA, tet-trpE^(fbr)DCBA strain to generate the ΔtrpRΔtnaA, tet-trpE^(fbr)DCBA, tet-aroG^(fbr)-serA strain. The tet-aroG^(fbr)-serA construct was further modified to encode a feedback resistant version of serA (serA^(fbr)). The newly generated tet-aroG^(fbr)-serA^(fbr) construct was used to produce the ΔtrpRΔtnaA, tet-trpE ^(fbr)DCBA, tet-aroG^(fbr)-serA^(fbr) strain, optimized to improve the rate of serine biosynthesis and maximize tryptophan production.

Example 41 Comparison of Various Tryptophan Producing Strains

Compare the rates of tryptophan production in the different strains generated, the following constructs and strains were generated according to methods and sequences described herein (e.g. Example 42), and assayed for tryptophan production in the presence of glucuronate as a carbon source under aerobic conditions. SYN2126 comprises ΔtrpRΔtnaA (ΔtrpRΔtnaA). SYN2323 comprises ΔtrpRΔtnaA and a tetracycline inducible construct for the expression of feedback resistant aroG on a plasmid (AtrpRΔtnaA, tet-aroGfbr). SYN2339 comprises ΔtrpRΔtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (ΔtrpRΔtnaA, tet-aroGfbr, tet-trpEfbrDCBA). SYN2473 comprises ΔtrpRΔtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG and SerA on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (ΔtrpRΔtnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA). SYN2476 comprises ΔtrpRΔtnaA and a tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a plasmid (AtrpRΔtnaA, tet-trpEfbrDCBA).

Overnight cultures were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Next, cells were induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm), spun down, washed with cmL M9, spun down again and resuspended in 3 mL M9+1% glucuronate. Cells were plated for CFU counting. For the assay, the cells were placed of 37 C with shaking at 250 rpm. Supernatants were collected at 1 h, 2 h, 3 h, 4 h 16 h for HPLC analysis for tryptophan. As seen in FIG. 42, results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production.

Example 42 Bacterial Production of Indole Acetic Acid (IAA)

The ability of a strain comprising tryptophan production circuits and additionally Indole-3-pyruvate decarboxylase from Enterobacter cloacae (IpdC) and Indole-3-acetaldehyde dehydrogenase from Ustilago maydis (Iadl) to produce indole acetic acid (IAA) was tested. The following strains were generated according to methods described herein and tested.

SYN2126: comprises ΔtrpR and ΔtnaA (ΔtrpRΔtnaA). SYN2339 comprises circuitry for the production of tryptophan; ΔtrpR and ΔtnaA, a first tetracline inducible trpEfbrDCBA construct on a first plasmid(pSC101), and a second tetracycline inducible aroGfbr construct on a second plasmid (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)) (FIG. 36B). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises trpDH-ipdC-iadl incorporated at the end of the second construct (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iad1 (p15A))(FIG. 39B).

Overnight cultures of the strains were diluted 1/100 in 3 mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Strains were then induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm). Cells were spun down, and resuspended in 1 mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 1 h, 2.5 h and 18 h for LCMS analysis of tryptophan and indole acetic acid as described herein.

As seen in FIG. 45, SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 containing the additional IAA producing circuitry produces amounts of IAA that are comparable to the amounts of tryptophan produced in its parent SYN2339. No tryptophan is measured, indicating that all tryptophan produced in SYN2342 is efficiently converted into IAA.

Example 43 Tryptamine Production Comparing Two Tryptophan Decarboxylases

The efficacy of two tryptophan decarboxylases (tdc), one from Catharanthus roseus (tdc_(Cr))and a second from Clostridium sporogenes (tdc_(Cs)) in producing tryptamine from tryptophan was tested. The following strains were generated according to methods described herein and tested.

SYN2339 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE^(thr)DCBA construct on a plasmid and another tetracycline inducible construct expressing aroG^(fbr) on a second plasmid (ΔtrpRΔtnaA, tetR-P_(tet)-trpE^(fbr)DCBA (pSC101), tetR-P_(tet)aroG^(fbr) (p15A)). SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine. SYN2340 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE^(thr)DCBA construct on a plasmid and another tetracycline inducible construct expressing aroG^(fbr) tdc_(Cr) on a second plasmid (ΔtrpRΔtnaA, tetR-P_(tet)-trpE^(fbr)DCBA (pSC101), tetR-P_(tet)aroG^(fbr)-tdc_(Cr) (p15A)). SYN2794 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE^(thr)DCBA construct on a plasmid and another tetracycline inducible construct expressing arod^(k) tdc_(Cs) on a second plasmid (ΔtrpRΔtnaA, tetR-P_(tet)-trpE^(fbr)DCBA (pSC101), tetR-P_(tet)-aroG^(fbr)-tdc_(Cs) (p15A)).

Overnight cultures of the strains were diluted 1/100 in 3 mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Strains were then induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm). Cells were spun down, and resuspended in 1 mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 3 h and 18 h for LCMS analysis of tryptophan and tryptamine, as described herein.

As seen in FIG. 46, Tdc_(Cs) from Clostridium sporogenes is more efficient than Tdc_(Cr) from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine

Example 44 Tryptophan and Anthranilic Acid Quantification in Bacterial Supernatant by LC-MS/MS

Tryptophan and Anthranilic acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 μL), and stored at −20° C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032m/mL) were prepared in water. Samples (10 μL) and standards were mixed with 90 μL of ACN/H20 (60:30, v/v) containing 1 μg/mL of Tryptophan-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000 rpm for 5 min. The solution (10 μL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well.

LC-MS/MS Method

Tryptophan and Anthranilic acid were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 60., Table 61, and Table 62 provide the summary of the LC-MS/MS method.

TABLE 60 HPLC Method Column Accucore aQ column, 2.6 μm (100 × 2.1 mm) Mobile Phase A 99.9% H2O, 0.1% Formic Acid Mobile Phase B 99.9% ACN, 0.1% Formic Acid Injection volume 10 uL

TABLE 61 HPLC Method: Flow Rate Time (min) (μL/min) A % B % −0.5 350 100 0 0.5 350 100 0 1.0 350 10 90 2.5 350 10 90 2.51 350 100 10

TABLE 62 Tandem Mass Spectrometry Ion Source HESI-II Polarity Positive SRM transitions Tryptophan 205.1/118.2 Anthranilic acid 138.1/92.2 Tryptophan-d5 210.1/151.1

Example 44 Quantification of Tryptamine in Bacterial Supernatant by Liquid Chromatography-Mass Spectrometry (LC-MS)

Tryptamine acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 μL), and stored at −20° C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 μm/mL) were prepared. Samples (10 μL) and standards were mixed with 90 μL of ACN/H2O (60:30, v/v) containing 1 μg/mL of tryptamine-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000 rpm for 5 min. The solution (10 μL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well.

LC-MS/MS Method

Tryptamine was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 63., Table 64, and Table 65 provide the summary of the LC-MS/MS method.

TABLE 63 HPLC Method Column Accucore aQ column, 2.6 μm (100 × 2.1 mm) Mobile Phase A 99.9% H2O, 0.1% Formic Acid Mobile Phase B 99.9% ACN, 0.1% Formic Acid Injection volume 10 uL

TABLE 64 HPLC Method: Flow Rate Time (min) (μL/min) A % B % −0.5 350 100 0 0.5 350 100 0 1.0 350 10 90 2.5 350 10 90 2.51 350 100 10

TABLE 65 Tandem Mass Spectrometry Ion Source HESI-II Polarity Positive SRM transitions Tryptamine 161.1/144.1

Example 45 Quantification of Tryptophan, Indole-3-acetate, Indole-3-lactate, Indole-3-propionate in Bacterial Supernatant by High-pressure Liquid Chromatography (HPLC)

Samples were thawed on ice and centrifuged at 3,220×g for 5min at 4° C. 80 μL of the supernatant was pipetted, mixed with 20 μL 0.5% formic acid in water, and analyzed by HPLC using a Shimadzu Prominence-I. HPLC conditions used for the quantification of tryptophan, indole-3-acetate, indole-3-lactate and indole-3-propionate are described in Table 66.

TABLE 66 HPLC Analysis Chromatography Calibration standards 250, 100, 20, 4, 0.8 μg/mL Column Luna 3 μm C18(2) 100 Å, 100 × 2 mm (catalog# 00D-4251-B0) Column Temperature 40° C. Injection Volume 10 μL Autosampler 10° C. Temperature Flow Rate 0.5 mL/min Mobile Phases A: water, 0.1% FA B: acetonitrile, 0.1% FA Time (min) % A % B Gradient 0 90 10 0.5 90 10 3 10 90 5 10 90 5.01 90 10 7 (end) Detection: Photodiode Array Detector (PDA) Polarity Positive Start Wavelength 190 nm End Wavelength 800 nm Spectrum resolution 512 Slit Width 8 nm Compound Wavelength (nm) Retention time (min) Tryptophan 274 1.3 Indole-3-acetate 274 3.5 Indole-lactate 274 3.3 Indole-3-propionate 274 3.7

Example 46 Generation of Constructs for Overproducing Therapeutic Molecules for Secretion

To produce strain capable of secreting anti-inflammatory or gut barrier enhancer polypeptides, e.g., GLP2, IL-22, IL-10 (viral or human), several constructs are designed employing different secretion strategies. Various GLP2, IL-22, IL-10 (viral or human) constructs are synthesized, and cloned into vector pBR322 for transformation of E. coli . In some embodiments, the constructs encoding the effector molecules are integrated into the genome. In some embodiments, the constructs encoding the effector molecules are on a plasmid, e.g., a medium copy plasmid. Table 67 lists exemplary polypeptide coding sequences used in the constructs.

TABLE 67 Polypeptide coding sequences Description Sequence SEQ ID NO GLP2 CATGCTGATGGTTCTTTCTCTGATGAGAT SEQ ID NO: 285 GAACACCATTCTTGATAATCTTGCCGCCA GGGACTTTATAAACTGGTTGATTCAGACC AAAATCACTGAC GLP2 codon CATGCTGACGGCTCTTTTTCTGACGAAAT SEQ ID NO: 286 optimized GAATACCATCCTGGATAATCTGGCGGCG CGTGATTTTATTAATTGGCTGATCCAAAC TAAAATTACTGATTAA FliC20-GLP2 ATGGCACAAGTCATTAATACCAACAGCC SEQ ID NO: 287 (F1iC20, start of FliC TCTCGCTGATCACTCAAAATAATATCAAC gene preceding AAGCATGCTGACGGCTCTTTTTCTGACGA GLP2 sequence AATGAATACCATCCTGGATAATCTGGCG underlined) GCGCGTGATTTTATTAATTGGCTGATCCA AACTAAAATTACTGATTAA GLP2 codon ATGCATGCTGACGGCTCTTTTTCTGACGA SEQ ID NO: 288 optimized (e.g., used AATGAATACCATCCTGGATAATCTGGCG in fliC construct) GCGCGTGATTTTATTAATTGGCTGATCCA AACTAAAATTACTGATTAA vIL10 codon ATGGGTACTGACCAATGTGATAATTTCCC SEQ ID NO: 289 optimized (e.g., used ACAAATGCTGCGTGATTTGCGCGACGCTT in fliC construct) TCTCGCGTGTGAAAACTTTTTTTCAGACT AAAGATGAGGTGGATAATCTGCTGCTGA AAGAGAGCCTGTTGGAAGATTTTAAAGG CTACTTGGGCTGTCAAGCGCTGTCGGAG ATGATTCAATTTTATCTGGAAGAGGTGAT GCCGCAAGCTGAGAACCAAGATCCGGAA GCGAAAGATCACGTGAATTCGCTGGGCG AGAATCTGAAAACTCTGCGTCTGCGTCTG CGTCGTTGTCACCGTTTTTTGCCGTGCGA AAACAAAAGTAAAGCTGTTGAGCAAATT AAAAACGCTTTTAACAAACTGCAGGAAA AAGGTATCTATAAAGCGATGAGCGAATT TGATATTTTTATTAATTATATTGAAGCTT ATATGACTATTAAAGCTCGCTAA vIL10 GGTACAGACCAATGTGACAATTTTCCCCA SEQ ID NO: 290 AATGTTGAGGGACCTAAGAGATGCCTTC AGTCGTGTTAAAACCTTTTTCCAGACAAA GGACGAGGTAGATAACCTTTTGCTCAAG GAGTCTCTGCTAGAGGACTTTAAGGGCT ACCTTGGATGCCAGGCCCTGTCAGAAAT GATCCAATTCTACCTGGAGGAAGTCATG CCACAGGCTGAAAACCAGGACCCTGAAG CCAAAGACCATGTCAATTCTTTGGGTGAA AATCTAAAGACCCTACGGCTCCGCCTGC GCCGTTGCCACAGGTTCCTGCCGTGTGAG AACAAGAGTAAAGCTGTGGAACAGATAA AAAATGCCTTTAACAAGCTGCAGGAAAA AGGAATTTACAAAGCCATGAGTGAATTT GACATTTTTATTAACTACATAGAAGCATA CATGACAATTAAAGCCAGG IL-22 codon GCACCGATCTCTTCCCACTGTCGCTTAGA SEQ ID NO: 291 optimized (e.g., use TAAATCGAATTTTCAACAACCTTATATTA with diffusible outer CGAATCGTACGTTTATGCTGGCTAAAGA membrane construct) AGCGTCATTAGCTGATAACAACACTGAT GTTCGCCTGATTGGTGAGAAATTGTTTCA CGGTGTGTCTATGTCAGAACGTTGCTACC TGATGAAACAAGTTCTGAATTTCACCCTG GAAGAAGTGTTGTTTCCGCAATCTGACCG CTTTCAACCGTATATGCAAGAGGTTGTGC CGTTTCTGGCGCGCCTGAGTAATCGCCTG AGCACTTGTCATATTGAGGGCGACGACC TGCATATTCAACGAAATGTTCAAAAATTG AAAGATACGGTGAAGAAACTGGGTGAAA GTGGTGAAATCAAAGCGATTGGTGAGCT GGATCTGCTGTTTATGTCATTGCGCAATG CGTGCATTTAA IL-22 codon ATGGCACCGATCTCTTCCCACTGTCGCTT SEQ ID NO: 292 optimized (e.g., used AGATAAATCGAATTTTCAACAACCTTATA in fliC construct) TTACGAATCGTACGTTTATGCTGGCTAAA GAAGCGTCATTAGCTGATAACAACACTG ATGTTCGCCTGATTGGTGAGAAATTGTTT CACGGTGTGTCTATGTCAGAACGTTGCTA CCTGATGAAACAAGTTCTGAATTTCACCC TGGAAGAAGTGTTGTTTCCGCAATCTGAC CGCTTTCAACCGTATATGCAAGAGGTTGT GCCGTTTCTGGCGCGCCTGAGTAATCGCC TGAGCACTTGTCATATTGAGGGCGACGA CCTGCATATTCAACGAAATGTTCAAAAAT TGAAAGATACGGTGAAGAAACTGGGTGA AAGTGGTGAAATCAAAGCGATTGGTGAG CTGGATCTGCTGTTTATGTCATTGCGCAA TGCGTGCATTTAA hIL-10 codon TCGCCAGGTCAAGGAACGCAGTCAGAGA SEQ ID NO: 293 optimized ATTCATGCACTCACTTTCCGGGCAATCTG CCGAATATGCTGCGCGATCTGCGAGATG CATTCTCTCGCGTGAAAACGTTCTTTCAA ATGAAAGATCAACTGGATAATCTGCTGC TGAAGGAGTCGTTGTTGGAGGATTTTAA GGGGTATCTGGGTTGTCAAGCACTGTCTG AAATGATTCAATTTTACTTGGAGGAAGTT ATGCCGCAAGCGGAAAACCAAGATCCGG ATATTAAGGCGCACGTGAACTCACTGGG CGAAAACCTGAAAACTTTGCGCCTGCGT CTGAGACGATGTCACCGATTCCTGCCGTG TGAAAACAAGTCAAAGGCGGTTGAGCAA GTTAAGAATGCTTTCAATAAGCTGCAAG AAAAGGGCATCTATAAAGCGATGTCTGA ATTTGATATCTTTATAAACTACATAGAAG CTTATATGACTATGAAGATTCGAAATTAA Monomerized hIL- TCGCCAGGTCAAGGAACGCAGTCAGAGA SEQ ID NO: 294 10 (codon opt) ATTCATGCACTCACTTTCCGGGCAATCTG CCGAATATGCTGCGCGATCTGCGAGATG CATTCTCTCGCGTGAAAACGTTCTTTCAA ATGAAAGATCAACTGGATAATCTGCTGC TGAAGGAGTCGTTGTTGGAGGATTTTAA GGGGTATCTGGGTTGTCAAGCACTGTCTG AAATGATTCAATTTTACTTGGAGGAAGTT ATGCCGCAAGCGGAAAACCAAGATCCGG ATATTAAGGCGCACGTGAACTCACTGGG CGAAAACCTGAAAACTTTGCGCCTGCGT CTGAGACGATGTCACCGATTCCTGCCGTG TGAAAACGGAGGAGGAAGTGGTGGTAAG TCAAAGGCGGTTGAGCAAGTTAAGAATG CTTTCAATAAGCTGCAAGAAAAGGGCAT CTATAAAGCGATGTCTGAATTTGATATCT TTATAAACTACATAGAAGCTTATATGACT ATGAAGATTCGAAATTAA

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, or SEQ ID NO: 294 or a functional fragment thereof.

Table 68 lists exemplary secretion tags, which can be added at the N-terminus when the diffusible outer membrane (DOM) method or the fliC method is used.

TABLE 68 Secretion Tags and FliC components Sequence Name Sequence SEQ ID NO fliC-FliC20 (e.g., used in GLP2 tgacggcgattgagccgacgggtggaaaccc SEQ ID NO: 295 construct) aaaacgtaatcaac GTGGGTACTC FliC20: start of the fliC gene CTTAAATTGGGTTCGAATGG which (in some constructs) ACC atggcacaagtcattaataccaacagc precedes the effector polypeptide ctctcgctgatcactcaaaataatatcaacaag sequence, see e.g., FIG. 30B and FIG. 30C shown in italics fliC: native fliC UTR in bold, optimized RBS underlined fliC-RBS (e.g., used in IL22 tgacggcgattgagccgacgggtggaaaccc SEQ ID NO: 296 construct) aaaacgtaatcaac tacgaacacttacagga fliC: native fliC UTR in bold, ggtaccca optimized RBS underlined fliC-RBS (e.g., used in GLP2 tgacggcgattgagccgacgggtggaaaccc SEQ ID NO: 297 construct) aaaacgtaatcaac aagtataaactctggga fliC: native fliC UTR in bold, ggttccta optimized RBS underlined fliC-RBS (e.g., used in vIL10 tgacggcgattgagccgacgggtggaaaccc SEQ ID NO: 298 construct) aaaacgtaatcaac tcaaatcc c ttaataagg fliC: native fliC UTR in bold, aggtaaa optimized RBS underlined RBS-phoA Ctctagaaataattttgtttaactttaagaaggaga SEQ ID NO: 299 RBS: underlined tatacatatgaaacaaagcactattgcactggca ctcttaccgttactgtttacccctgtgacaaaagc g phoA atgaaacaaagcactattgcactggcactcttac SEQ ID NO: 300 cgttactgtttacccctgtgacaaaagcg RBS-ompF Ctctagaaataattttgtttaactttaagaaggaga SEQ ID NO: 301 RBS: underlined tatacatatgatgaagcgcaatattctggcagtga tcgtccctgctctgttagtagcaggtactgcaaac gct ompF atgatgaagcgcaatattctggcagtgatcgtcc SEQ ID NO: 302 ctgctctgttagtagcaggtactgcaaacgct RBS-cvaC Ctctagaaataattttgtttaactttaagaaggaga SEQ ID NO: 303 RBS: underlined tatacatATGAGAACTCTGACTCT AAATGAATTAGATTCTGTTTC TGGTGGT cvaC ATGAGAACTCTGACTCTAAAT SEQ ID NO: 304 GAATTAGATTCTGTTTCTGGT GGT RBS-phoA (Optimized, e.g., GACGCCAGAGAGTTAAGGGG SEQ ID NO: 305 used in IL10 construct) GTTAAATGAAACAATCGACC RBS: underlined ATCGCATTGGCGCTGCTTCCT CTATTGTTCACACCGGTGACA AAGGCA Optimized phoA ATGAAACAATCGACCATCGC SEQ ID NO: 306 ATTGGCGCTGCTTCCTCTATT GTTCACACCGGTGACAAAGG CA RBS-TorA ctctagaaataattttgtttaactttaagaaggagat SEQ ID NO: 307 RBS: underlined atacatATGAACAATAACGATCT CTTTCAGGCATCACGTCGGCG TTTTCTGGCACAACTCGGCGG CTTAACCGTCGCCGGGATGCT GGGGCCGTCATTGTTAACGCC GCGACGTGCGACTGCG TorA ATGAACAATAACGATCTCTTT SEQ ID NO: 308 CAGGCATCACGTCGGCGTTTT CTGGCACAACTCGGCGGCTTA ACCGTCGCCGGGATGCTGGG GCCGTCATTGTTAACGCCGCG ACGTGCGACTGCG RBS-TorA alternate CCCACATTCGAGGTACTAAatg SEQ ID NO: 309 aacaataacgatctctttcaggcatcacgtcggc gttttctggcacaactcggcggcttaaccgtcgc cgggatgctggggacgtcattgttaacgccgcg ccgtgcgactgcggcgcaagcggcg TorA (alternate) atgaacaataacgatctctttcaggcatcacgtcg SEQ ID NO: 310 gcgttttctggcacaactcggcggcttaaccgtc gccgggatgctggggacgtcattgttaacgccg cgccgtgcgactgcggcgcaagcggcg RBS-fdnG ACCCTATTACACACCTAAGGA SEQ ID NO: 311 GGCCAAATACatggacgtcagtcgcag acaattttttaaaatctgcgcgggcggtatggcg ggaacaacagtagcagcattgggctttgccccg aagcaagcactggct fdnG atggacgtcagtcgcagacaattttttaaaatctg SEQ ID NO: 312 cgcgggcggtatggcgggaacaacagtagca gcattgggctttgccccgaagcaagcactggct RBS-dmsA TACGCAAAAAACATAATTTAA SEQ ID NO: 313 GAGAGGATAAACatgaaaacgaaaa tccctgatgcggtattggctgctgaggtgagtcg ccgtggtttggtaaaaacgacagcgatcggcgg cctggcaatggccagcagcgcattaacattacct tttagtcggattgcgcacgct dmsA atgaaaacgaaaatccctgatgcggtattggctg SEQ ID NO: 314 ctgaggtgagtcgccgtggtttggtaaaaacgac agcgatcggcggcctggcaatggccagcagcg cattaacattaccttttagtcggattgcgcacgct

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, and SEQ ID NO: 313. Table 69 lists exemplary promoter sequences and miscellaneous construct sequences.

TABLE 69 Promoter Sequences and Various Construct Sequences SEQ ID Description Sequence NO TetR/TetA gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaag SEQ ID Promoter gccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaata NO: 315 atggcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgacttgatc ttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattctct agtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttct gtaggccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaa acttttagcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagtgag tatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttttacat gccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaacctt cgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctaga catcattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatc agtgatagagaaaagtgaa fliC Promoter agcgggaataaggggcagagaaaagagtatttcgtcgactaacaaaaaatggctgtttgt SEQ ID gaaaaaaattctaaaggttgttttacgacagacgataacagggt NO: 316 FnrS ggtaccAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT SEQ ID Promoter AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAA NO: 317 CGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGG CAATATCTCTCTTggatcc DOM cacatttccccgaaaagtgccgatggccccccgatggtagtgtggcccatgcgagagtagg SEQ ID Construct gaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttat NO: 318 Terminator ctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacg ttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcat caaattaagcagaaggccatcctgacggatggcctttttgcgtggccagtgccaagcttgc atgcagattgcagcattacacgtcttgagcgattgtgtaggctggagctgcttc FRT Site gaagttcctatactttctagagaataggaacttcggaataggaacttc SEQ ID NO: 319 Kanamycin aagatcccctcacgctgccgcaagcactcagggcgcaagggctgctaaaggaagcggaa SEQ ID Resistance cacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggc NO: 320 Cassette (for tatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggctta integration catggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctg in between gggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgcc FRT sites) aaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcg catgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattc ggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcag cgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcag gacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcg acgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatc tcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagc gagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatc aggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgag gatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgctt ttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttgg ctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttac ggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctga gcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggc tggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccagcttcaaaag cgctct

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, and SEQ ID NO: 320. Table 70 Lists exemplary secretion constructs.

TABLE 70 Non-limiting Examples of Secretion Constructs Description Sequence SEQ ID NO: FliC20-glp2; a human cgttccttgtagggcgtcatagcgttcgacggcattaagtaacccaatgcc SEQ ID NO: GLP2 construct gcccgcctgtagcagatcgtcaagttccacgctcgcgggcagtcgaacct 321 inserted into the FliC gcaggcgcaatgcttcgtgacgcaccagcgggacataacgctgccacag locus, under the cgagtgtttatccattacaccttcagcggtatagagtgaattcacgataaaca control of the native gccctgcgttatatgagttatcggcatgattatccgtttctgcagggtattaat FliC promoter cggacgattagtgggtgaaatgaggggttatttgggggttaccggtaaatt gcgggcagaaaaaaccccgccgttggcggggaagcacgttgctggcaa attaccattcatgttgccggatgcggcgtaaacgccttatccggcctacaaa aatgtgcaaattcaataaattgcaattccccttgtaggcctgataagcgcag cgcatcaggcaatttggcgttgccgtcagtctcagttaatcaggttacggcg attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG cattaggaacctcccagagtttatacttgttgattacgttttgggtttccaccc gtcggctcaatcgccgtcaaccctgttatcgtctgtcgtaaaacaacctttag aatttttttcacaaacagccattttttgttagtcgacgaaatactcttttctctgc cccttattcccgctattaaaaaaaacaattaaacgtaaactttgcgcaattca ggccgataaccccggtattcgttttacgtgtcgaaagataaaCGAAGT TCCTATACTTTCTAGAGAATAGGAACTTCGG AATAGGAACTTCATTTctcgttcgctgccacctaagaatact ctacggtcacatacAAATGGCGCGCCTTACGCCCCGC CCTGCCACTCATCGCAGTACTGTTGTATTCAT TAAGCATCTGCCGACATGGAAGCCATCACAA ACGGCATGATGAACCTGAATCGCCAGCGGCA TCAGCACCTTGTCGCCTTGCGTATAATATTTG CCCATGGTGAAAACGGGGGCGAAGAAGTTGT CCATATTGGCCACGTTTAAATCAAAACTGGT GAAACTCACCCAGGGATTGGCTGAGACGAAA AACATATTCTCAATAAACCCTTTAGGGAAAT AGGCCAGGTTTTCACCGTAACACGCCACATC TTGCGAATATATGTGTAGAAACTGCCGGAAA TCGTCGTGGTATTCACTCCAGAGCGATGAAA ACGTTTCAGTTTGCTCATGGAAAACGGTGTA ACAAGGGTGAACACTATCCCATATCACCAGC TCACCGTCTTTCATTGCCATACGTAATTCCGG ATGAGCATTCATCAGGCGGGCAAGAATGTGA ATAAAGGCCGGATAAAACTTGTGCTTATTTTT CTTTACGGTCTTTAAAAAGGCCGTAATATCC AGCTGAACGGTCTGGTTATAGGTACATTGAG CAACTGACTGAAATGCCTCAAAATGTTCTTT ACGATGCCATTGGGATATATCAACGGTGGTA TATCCAGTGATTTTTTTCTCCATTTTAGCTTCC TTAGCTCCTGAAAATCTCGACAACTCAAAAA ATACGCCCGGTAGTGATCTTATTTCATTATGG TGAAAGTTGGAACCTCTTACGTGCCGATCAA CGTCTCATTTTCGCCAAAAGTTGGCCCAGGG CTTCCCGGTATCAACAGGGACACCAGGATTT ATTTATTCTGCGAAGTGATCTTCCGTCACAGG TAGGCGCGCCGAAGTTCCTATACTTTCTAGA GAATAGGAACTTCGGAATAGGAACTctcaccgcc gcgcaaaaagcgacgctaacccctatttcaaatcagcaatcgtcgtttacc gctaaacttagcgcctacggtacgctgaaaagcgcgctgacgactttcca gaccgccaatactgcattgtctaaagccgatcttttttccgctaccagcacc accagcagcaccaccgcgttcagtgccaccaccgcgggtaatgccatcg ccgggaaatacaccatcagcgtcacccatctggcgcaggcgcaaaccct gacaacgcgcaccaccagagacgatacgaaaacggcgatcgccacca gcgacagcaaactcaccattcaacaaggcggcgacaaagatccgatttcc attgatatcagcgcggctaactcgtctttaagcgggatccgtgatgccatca acaacgcaaaagcaggcgtaagcgcaagcatcattaacgtgggtaacgg tgaatatcgtctgtcagtcacatcaaatgacaccggcct FliC20 with optimized attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc SEQ ID NO: RBS-GLP2 and UTR- agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG 322 FliC cattaggaacctcccagagtttatacttgttgattacgttttgggtttccaccc gtcggctcaatcgccgtca human GLP2 cgttccttgtagggcgtcatagcgttcgacggcattaagtaacccaatgcc SEQ ID NO: construct,, including gcccgcctgtagcagatcgtcaagttccacgctcgcgggcagtcgaacct 323 the N terminal 20 gcaggcgcaatgcttcgtgacgcaccagcgggacataacgctgccacag amino acids of FliC cgagtgtttatccattacaccttcagcggtatagagtgaattcacgataaaca (reverse orientation), gccctgcgttatatgagttatcggcatgattatccgtttctgcagggtattaat inserted into the FliC cggacgattagtgggtgaaatgaggggttatttgggggttaccggtaaatt locus under the control gcgggcagaaaaaaccccgccgttggcggggaagcacgttgctggcaa of a tet inducible attaccattcatgttgccggatgcggcgtaaacgccttatccggcctacaaa promoter, with TetR aatgtgcaaattcaataaattgcaattccccttgtaggcctgataagcgcag and chloramphenicol cgcatcaggcaatttggcgttgccgtcagtctcagttaatcaggttacggcg resistance. attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG cttgttgatattattttgagtgatcagcgagaggctgttggtattaatgacttgt gccatGGTCCATTCGAACCCAATTTAAGGAGTA CCCACgttgattacgttttgggtttccacccgtcggctcaatcgccgtca ttctctatcactgatagggagtggtaaaataactctatcaatgatagagtgtc aacaaaaattaggaattaatgatgtctagattagataaaagtaaagtgattaa cagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccg taaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgt aaaaaataagcgggctttgctcgacgccttagccattgagatgttagatag gcaccatactcacttttgccctttagaaggggaaagctggcaagattttttac gtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagca aaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaa aatcaattagccatttatgccaacaaggtttttcactagagaatgcattatatg cactcagcgctgtggggcattttactttaggttgcgtattggaagatcaaga gcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgc cgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagcc agccttcttattcggccttgaattgatcatatgcggattagaaaaacaactta aatgtgaaagtgggtcttaagaattatttcacaaacagccattattgttagtc gacgaaatactcttttctctgccccttattcccgctattaaaaaaaacaattaa acgtaaactttgcgcaattcaggccgataaccccggtattcgttttacgtgtc gaaagataaaCGAAGTTCCTATACTTTCTAGAGAA TAGGAACTTCGGAATAGGAACTTCATTTctcgtt cgctgccacctaagaatactctacggtcacatacAAATGGCGCG CCTTACGCCCCGCCCTGCCACTCATCGCAGTA CTGTTGTATTCATTAAGCATCTGCCGACATGG AAGCCATCACAAACGGCATGATGAACCTGAA TCGCCAGCGGCATCAGCACCTTGTCGCCTTG CGTATAATATTTGCCCATGGTGAAAACGGGG GCGAAGAAGTTGTCCATATTGGCCACGTTTA AATCAAAACTGGTGAAACTCACCCAGGGATT GGCTGAGACGAAAAACATATTCTCAATAAAC CCTTTAGGGAAATAGGCCAGGTTTTCACCGT AACACGCCACATCTTGCGAATATATGTGTAG AAACTGCCGGAAATCGTCGTGGTATTCACTC CAGAGCGATGAAAACGTTTCAGTTTGCTCAT GGAAAACGGTGTAACAAGGGTGAACACTATC CCATATCACCAGCTCACCGTCTTTCATTGCCA TACGTAATTCCGGATGAGCATTCATCAGGCG GGCAAGAATGTGAATAAAGGCCGGATAAAA CTTGTGCTTATTTTTCTTTACGGTCTTTAAAA AGGCCGTAATATCCAGCTGAACGGTCTGGTT ATAGGTACATTGAGCAACTGACTGAAATGCC TCAAAATGTTCTTTACGATGCCATTGGGATAT ATCAACGGTGGTATATCCAGTGATTTTTTTCT CCATTTTAGCTTCCTTAGCTCCTGAAAATCTC GACAACTCAAAAAATACGCCCGGTAGTGATC TTATTTCATTATGGTGAAAGTTGGAACCTCTT ACGTGCCGATCAACGTCTCATTTTCGCCAAA AGTTGGCCCAGGGCTTCCCGGTATCAACAGG GACACCAGGATTTATTTATTCTGCGAAGTGA TCTTCCGTCACAGGTAGGCGCGCCGAAGTTC CTATACTTTCTAGAGAATAGGAACTTCGGAA TAGGAACTctcaccgccgcgcaaaaagcgacgctaacccctattt caaatcagcaatcgtcgtttaccgctaaacttagcgcctacggtacgctga aaagcgcgctgacgactttccagaccgccaatactgcattgtctaaagccg atcttttttccgctaccagcaccaccagcagcaccaccgcgttcagtgcca ccaccgcgggtaatgccatcgccgggaaatacaccatcagcgtcaccca tctggcgcaggcgcaaaccctgacaacgcgcaccaccagagacgatac gaaaacggcgatcgccaccagcgacagcaaactcaccattcaacaagg cggcgacaaagatccgatttccattgatatcagcgcggctaactcgtcttta agcgggatccgtgatgccatcaacaacgcaaaagcaggcgtaagcgca agcatcattaacgtgggtaacggtgaatatcgtctgtcagtcacatcaaatg acaccggcct human GLP2 ttaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcca SEQ ID NO: construct,, including gattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATGc 324 the N terminal 20 ttgttgatattattttgagtgatcagcgagaggctgttggtattaatgacttgtg amino acids of FliC ccat (reverse orientation) human GLP2 ttaagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaa SEQ ID NO: construct with a N ggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatag 325 terminal OmpF cttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt secretion tag (sec- agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgcccc dependent secretion acagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata system) under the aaaaggctaattgattttcgagagtttcatactgatttctgtaggccgtgtacc control of a tet taaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaactttt inducible promoter, agcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagt includes TetR in gagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagccc reverse direction gcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaat gcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt gttgacactctatcattgatagagttattttaccactccctatcagtgatagagaa aagtgaactctagaaataattttgtttaactttaagaaggagatatacatatga tgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcaggtac tgcaaacgctcatgctgatggttctttctctgatgagatgaacaccattcttga taatcttgccgccagggactttataaactggttgattcagaccaaaatcactg acaggtgacacatttccccgaaaagtgccgatggccccccgatggtagtg tggccccatgcgagagtagggaactgccaggcatcaaataaaacgaaag gctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgct ctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagca acggcccggagggtggcgggcaggacgcccgccataaactgccaggc atcaaattaagcagaaggccatcctgacggatggcctttttgcgtggccag tgccaagcttgcatgcagattgcagcattacacgtcttgagcgattgtgtag gctggagctgcttcgaagttcctatactttctagagaataggaacttcggaat aggaacttc human GLP2 atgatgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcag SEQ ID NO: construct with a N gtactgcaaacgctcatgctgatggttctttctctgatgagatgaacaccatt 326 terminal OmpF cttgataatcttgccgccagggactttataaactggttgattcagaccaaaat secretion tag (sec- cactgacaggtga dependent secretion system) human GLP2 taagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaa SEQ ID NO: construct with a N ggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatag 327 terminal TorA cttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt secretion tag (tat agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgcccc secretion system) acagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata under the control of aaaaggctaattgattttcgagagtttcatactgatttctgtaggccgtgtacc a tet inducible taaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaactttt promoter agcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagt gagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagccc gcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaat gcgctgttaatcactttacttttatctaatctagacatcattaattcctaattttt gttgacactctatcattgatagagttattttaccactccctatcagtgatagagaa aagtgaactctagaaataattttgtttaactttaagaaggagatatacatAT GAACAATAACGATCTCTTTCAGGCATCACGT CGGCGTTTTCTGGCACAACTCGGCGGCTTAA CCGTCGCCGGGATGCTGGGGCCGTCATTGTT AACGCCGCGACGTGCGACTGCGcatgctgatggttctt tctctgatgagatgaacaccattcttgataatcttgccgccagggactttata aactggttgattcagaccaaaatcactgactaataacacatttccccgaaaa gtgccgatggccccccgatggtagtgtggcccatgcgagagtagggaac tgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttc gttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccg ggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggca ggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcc tgacggatggcctttttgcgtggccagtgccaagcttgcatgcagattgca gcattacacgtcttgagcgattgtgtaggctggagctgcttcgaagttcctat actttctagagaataggaacttcggaataggaacttc GLP-2 with TORA tag ATGAACAATAACGATCTCTTTCAGGCATCAC SEQ ID NO: GTCGGCGTTTTCTGGCACAACTCGGCGGCTT 328 AACCGTCGCCGGGATGCTGGGGCCGTCATTG TTAACGCCGCGACGTGCGACTGCGcatgctgatggt tctttctctgatgagatgaacaccattcttgataatcttgccgccagggacttt ataaactggttgattcagaccaaaatcactgac

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQ ID NO: 328. Table 71 lists exemplary secretion constructs.

TABLE 71 Non-limiting Examples of Secretion Constructs Description Sequences SEQ ID NO Ptet-phoA-hIL10 gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat SEQ ID NO: gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca 329 aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt gatagagttattttaccactccctatcagtgatagagaaaagtgaa GACGCCAGAGAGTTAAGGGGGTTAAATGAA ACAATCGACCATCGCATTGGCGCTGCTTCCTC TATTGTTCACACCGGTGACAAAGGCA TCGCCAGGTCAAGGAACGCAGTCAGAGAATT CATGCACTCACTTTCCGGGCAATCTGCCGAA TATGCTGCGCGATCTGCGAGATGCATTCTCTC GCGTGAAAACGTTCTTTCAAATGAAAGATCA ACTGGATAATCTGCTGCTGAAGGAGTCGTTG TTGGAGGATTTTAAGGGGTATCTGGGTTGTC AAGCACTGTCTGAAATGATTCAATTTTACTTG GAGGAAGTTATGCCGCAAGCGGAAAACCAA GATCCGGATATTAAGGCGCACGTGAACTCAC TGGGCGAAAACCTGAAAACTTTGCGCCTGCG TCTGAGACGATGTCACCGATTCCTGCCGTGT GAAAACAAGTCAAAGGCGGTTGAGCAAGTT AAGAATGCTTTCAATAAGCTGCAAGAAAAGG GCATCTATAAAGCGATGTCTGAATTTGATAT CTTTATAAACTACATAGAAGCTTATATGACT ATGAAGATTCGAAATTAA phoA-hIL10 GACGCCAGAGAGTTAAGGGGGTTAAATGAA SEQ ID NO: ACAATCGACCATCGCATTGGCGCTGCTTCCTC 330 TATTGTTCACACCGGTGACAAAGGCA TCGCCAGGTCAAGGAACGCAGTCAGAGAATT CATGCACTCACTTTCCGGGCAATCTGCCGAA TATGCTGCGCGATCTGCGAGATGCATTCTCTC GCGTGAAAACGTTCTTTCAAATGAAAGATCA ACTGGATAATCTGCTGCTGAAGGAGTCGTTG TTGGAGGATTTTAAGGGGTATCTGGGTTGTC AAGCACTGTCTGAAATGATTCAATTTTACTTG GAGGAAGTTATGCCGCAAGCGGAAAACCAA GATCCGGATATTAAGGCGCACGTGAACTCAC TGGGCGAAAACCTGAAAACTTTGCGCCTGCG TCTGAGACGATGTCACCGATTCCTGCCGTGT GAAAACAAGTCAAAGGCGGTTGAGCAAGTT AAGAATGCTTTCAATAAGCTGCAAGAAAAGG GCATCTATAAAGCGATGTCTGAATTTGATAT CTTTATAAACTACATAGAAGCTTATATGACT ATGAAGATTCGAAATTAA fliC UTR-RBS- tgacggcgattgagccgacgggtggaaacccaaaacgtaatcaac t SEQ ID NO: pvIL10 caaatcccttaataaggaggtaaa ATGGGTACTGACCAA 331 TGTGATAATTTCCCACAAATGCTGCGTGATTT GCGCGACGCTTTCTCGCGTGTGAAAACTTTTT TTCAGACTAAAGATGAGGTGGATAATCTGCT GCTGAAAGAGAGCCTGTTGGAAGATTTTAAA GGCTACTTGGGCTGTCAAGCGCTGTCGGAGA TGATTCAATTTTATCTGGAAGAGGTGATGCC GCAAGCTGAGAACCAAGATCCGGAAGCGAA AGATCACGTGAATTCGCTGGGCGAGAATCTG AAAACTCTGCGTCTGCGTCTGCGTCGTTGTCA CCGTTTTTTGCCGTGCGAAAACAAAAGTAAA GCTGTTGAGCAAATTAAAAACGCTTTTAACA AACTGCAGGAAAAAGGTATCTATAAAGCGAT GAGCGAATTTGATATTTTTATTAATTATATTG AAGCTTATATGACTATTAAAGCTCGCTAA Ptet-phoA-vIL10 Gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat SEQ ID NO: gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca 332 aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt gatagagttattttaccactccctatcagtgatagagaaaagtgaa GACGCCAGAGAGTTAAGGGGGTTAAATGAA ACAATCGACCATCGCATTGGCGCTGCTTCCTC TATTGTTCACACCGGTGACAAAGGCA GGTACAGACCAATGTGACAATTTTCCCCAAA TGTTGAGGGACCTAAGAGATGCCTTCAGTCG TGTTAAAACCTTTTTCCAGACAAAGGACGAG GTAGATAACCTTTTGCTCAAGGAGTCTCTGCT AGAGGACTTTAAGGGCTACCTTGGATGCCAG GCCCTGTCAGAAATGATCCAATTCTACCTGG AGGAAGTCATGCCACAGGCTGAAAACCAGG ACCCTGAAGCCAAAGACCATGTCAATTCTTT GGGTGAAAATCTAAAGACCCTACGGCTCCGC CTGCGCCGTTGCCACAGGTTCCTGCCGTGTG AGAACAAGAGTAAAGCTGTGGAACAGATAA AAAATGCCTTTAACAAGCTGCAGGAAAAAGG AATTTACAAAGCCATGAGTGAATTTGACATT TTTATTAACTACATAGAAGCATACATGACAA TTAAAGCCAGG phoA-vIL10 GACGCCAGAGAGTTAAGGGGGTTAAATGAA SEQ ID NO: ACAATCGACCATCGCATTGGCGCTGCTTCCTC 333 TATTGTTCACACCGGTGACAAAGGCA GGTACAGACCAATGTGACAATTTTCCCCAAA TGTTGAGGGACCTAAGAGATGCCTTCAGTCG TGTTAAAACCTTTTTCCAGACAAAGGACGAG GTAGATAACCTTTTGCTCAAGGAGTCTCTGCT AGAGGACTTTAAGGGCTACCTTGGATGCCAG GCCCTGTCAGAAATGATCCAATTCTACCTGG AGGAAGTCATGCCACAGGCTGAAAACCAGG ACCCTGAAGCCAAAGACCATGTCAATTCTTT GGGTGAAAATCTAAAGACCCTACGGCTCCGC CTGCGCCGTTGCCACAGGTTCCTGCCGTGTG AGAACAAGAGTAAAGCTGTGGAACAGATAA AAAATGCCTTTAACAAGCTGCAGGAAAAAGG AATTTACAAAGCCATGAGTGAATTTGACATT TTTATTAACTACATAGAAGCATACATGACAA TTAAAGCCAGG Ptet-PhoA-IL22 Gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat SEQ ID NO: gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca 334 aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt gatagagttattttaccactccctatcagtgatagagaaaagtgaa GACGCCAGAGAGTTAAGGGGGTTAAATGAA ACAATCGACCATCGCATTGGCGCTGCTTCCTC TATTGTTCACACCGGTGACAAAGGCA GCACCGATCTCTTCCCACTGTCGCTTAGATAA ATCGAATTTTCAACAACCTTATATTACGAATC GTACGTTTATGCTGGCTAAAGAAGCGTCATT AGCTGATAACAACACTGATGTTCGCCTGATT GGTGAGAAATTGTTTCACGGTGTGTCTATGTC AGAACGTTGCTACCTGATGAAACAAGTTCTG AATTTCACCCTGGAAGAAGTGTTGTTTCCGC AATCTGACCGCTTTCAACCGTATATGCAAGA GGTTGTGCCGTTTCTGGCGCGCCTGAGTAATC GCCTGAGCACTTGTCATATTGAGGGCGACGA CCTGCATATTCAACGAAATGTTCAAAAATTG AAAGATACGGTGAAGAAACTGGGTGAAAGT GGTGAAATCAAAGCGATTGGTGAGCTGGATC TGCTGTTTATGTCATTGCGCAATGCGTGCATT TAA PhoA-IL22 GACGCCAGAGAGTTAAGGGGGTTAAATGAA SEQ ID NO: ACAATCGACCATCGCATTGGCGCTGCTTCCTC 335 TATTGTTCACACCGGTGACAAAGGCA GCACCGATCTCTTCCCACTGTCGCTTAGATAA ATCGAATTTTCAACAACCTTATATTACGAATC GTACGTTTATGCTGGCTAAAGAAGCGTCATT AGCTGATAACAACACTGATGTTCGCCTGATT GGTGAGAAATTGTTTCACGGTGTGTCTATGTC AGAACGTTGCTACCTGATGAAACAAGTTCTG AATTTCACCCTGGAAGAAGTGTTGTTTCCGC AATCTGACCGCTTTCAACCGTATATGCAAGA GGTTGTGCCGTTTCTGGCGCGCCTGAGTAATC GCCTGAGCACTTGTCATATTGAGGGCGACGA CCTGCATATTCAACGAAATGTTCAAAAATTG AAAGATACGGTGAAGAAACTGGGTGAAAGT GGTGAAATCAAAGCGATTGGTGAGCTGGATC TGCTGTTTATGTCATTGCGCAATGCGTGCATT TAA GACGCCAGAGAGTTAAGGGGGTTAAATGAA SEQ ID NO: ACAATCGACCATCGCATTGGCGCTGCTTCCTC 336 TATTGTTCACACCGGTGACAAAGGCA

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, and SEQ ID NO: 336.

Example 47 Bacterial Secretion of hIL-10 and vIL-10

To determine whether the human IL-10 and vIL-10 expressed by engineered bacteria is secreted, the concentration of IL-10 in the bacterial supernatant from a selection of engineered strains comprising various hIL-10 and vIL-10 constructs/strains was measured (see Table 62, Table 63, Table 64, Table 65, Table 66 for components and sequences for hIL-10 and vIL-10 constructs/strains).

E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4° C. then placed on ice until further processing.

The concentration of hIL-10 in the cell-free medium and in the bacterial cell extract was measured by hIL-10 ELISA (R&D Systems DY217B), according to manufacturer's instructions Similarly, to determine the concentrations of vIL-10 an Ultrasensitive ELISA kit (Alpco, 45-I10HUU-E01) was employed using commercially available recombinant vIL-10 (R&D Systems, 915-VL-010). All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-10. Standard curves were generated using both human and viral recombinant proteins. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Table 72 and Table 73 summarize levels of hIL10 and vIL-10 measured in the supernatant. The data show that both vIL-10 and hIL-10 are secreted at various levels from the different bacterial strains.

TABLE 72 hIL-10 Secretion hu IL-10 (ng/ml) Sample (extracellular) WT 0 IL-10 Plasmid (Nissle 8.4 pUC57.Ptet-phoA-hIL10) IL-10 plasmid/lpp 19.3 (lpp::Cm pUC57.Ptet-phoA- hIL10) 2083 IL-10 plasmid/nlpI 20.5 (nlpI::Cm pUC57.Ptet-phoA- hIL10) 2084 IL-10 plasmid/tolA 21.4 (tolA::Cm pUC57.Ptet-phoA- hIL10) 2085 IL-10 plasmid/pal 28.4 (PAL::Cm pUC57.Ptet-phoA- hIL10)

TABLE 73 vIL-10 Secretion vIL-10 (ng/ml) Sample (extracellular) WT 0 fliC-pvIL10 (Nissle 29 pUN fli-vIL10 Kan Cm) fliC::vIL10 (Nissle 9 fliC::vIL10 delta fliD CmR) vIL-10 lpp (Nissle lpp 527 mutant with vIL10 pBR3222 tet plasmid) vIL-10 nlp1 (Nissle 982 delta nlpI::CmR pBR322.Ptet- phoA-vIL10) vIL-10 tolA (Nissle 428 delta tolA::CmR pBR322.Ptet- phoA-vIL10) vIL-10 pal (Nissle delta 1090 PAL::CmR pBR322.Ptet- phoA-vIL10

Co-Culture Studies

To determine whether the hIL-10 and viral IL-10 expressed by the genetically engineered bacteria shown in Table 72 and Table 73 is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant containing secreted human or viral IL-10 is added to the growth medium of a Raji cells (a hematopoietic cell line) and J774a1 cells (a macrophage cell line). IL-10 is known to induce the phosphorylation of STAT3 in these cells Functional activity of bacterially secreted IL-10 is therefore assessed by its ability to phosphorylate STAT3 in Raji and J774a1 cells.

Raji cells are grown in RPM! 1640 supplemented with 10% FBS supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, RPMI 1640 supplemented with 10% FBS (1e6/24 well) are serum starved overnight. Titrations of either recombinant human IL-10 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for 30 minutes. Cells are harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) is run in triplicate for all samples, according to manufacturer's instructions. PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity.

Competition Studies

As an additional control for specificity, a competition assay is performed. Titrations of anti-IL10 antibody are pre-incubated with constant concentrations of either rhIL22 (data not shown) or supernatants from the engineered bacteria expressing human or viral IL-22 for 15 min. Next, the supernatants/ rhIL2 solutions are added to serum-starved Raji cells (1e6/well) and cells are incubated for 30 min followed by pSTAT3 ELISA as described above.

In other embodiments, similar studies are conducted with J774a1 cells.

Example 48 Bacterial Secretion of GLP-2

To determine whether the human GLP-2 expressed by engineered bacteria is secreted, the concentration of GLP-2 in the bacterial supernatant from two engineered strains comprising GLP-2 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing GLP-2 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing GLP-2, further comprising a deletion in degP (see Table 74).

E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4° C. then placed on ice until further processing.

The concentration of GLP-2 in the cell-free medium and in the bacterial cell extract was measured by Human GLP2 ELISA Kit (Competitive EIA) (LSBio), according to manufacturer's instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of GLP-2. Standard curves were generated using recombinant GLP-2. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. As seen in Table 74, deletion of degP, a periplasmic protease, improved secretion levels over 3-fold.

TABLE 74 GLP-2 Secretion DOM mut ng/ml WT 1.14 PAL::CmR Ptet-ompF-GLP2 1793.2 PAL::CmR ompT::Kan Ptet- 1142.1 ompF-GLP2 PAL::CmR ompT::Kan phoA- 5360.4 GLP2 fusion

Co-Culture Studies

To determine whether the hGLP-2 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant (from both strains shown above) containing secreted human GLP-2 is added to the growth medium of Caco-2 cells and CCD-18 Co cells. The Caco-2 cell line is a continuous cell of heterogeneous human epithelial colorectal adenocarcinoma cells. As described e.g., in Jasleen et al. (Dig Dis Sci. 2002 May; 47(5):1135-40) GLP-2 stimulates proliferation and [3H]thymidine incorporation in Caco-2 and T84 cells. Additionally, GLP-2 stimulates VEGFA secretion in these cells (see., e.g., Bulut et al, Eur J Pharmacol. 2008 Jan. 14; 578(2-3):279-85.

Functional activity of bacterially secreted GLP-2 is therefore assessed by its ability to induce proliferation and VEGF secretion.

Caco-2 are grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Caco-2 cells (1e6/24 well) are serum starved overnight. Titrations of either recombinant human GLP-2 (50 and 250 nM) diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for a defined time.

For cell proliferation assays, cells are harvested and resuspended in lysis buffer. The cells are assayed after 12, 24, 48, and 72 hours of incubation. Cell proliferation is measured using a Cell proliferation assay kit according to manufacturer's instruction (e.g., a Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay).

For the measurements of VEFA secretion, cells are harvested and resuspended in lysis buffer, and concentrations of GLP-2 in the medium are determined ELISA

PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity.

Competition Studies

As an additional control for specificity, a competition assay is performed. Titrations of anti-GLP-2 antibody are pre-incubated with constant concentrations of either recombinant GLP-2 or supernatants from the engineered bacteria for 15 min. Next, the supernatants/ rhIL2 solutions are added to serum-starved Cac-2 cells (1e6/well) and cells are incubated for 30 min followed by VEGFA ELISA as described above.

Example 49 Bacterial Secretion of IL-22

To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant from two engineered strains comprising IL-22 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing IL-22 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing IL-22, further comprising a deletion in degP (Table 75).

E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4.0 then placed on ice until further processing.

The concentration of IL-22 in the cell-free medium and in the bacterial cell extract was measured by hIL-22 ELISA (R&D Systems (DY782) ELISA for hIL-22), according to manufacturer's instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Standard curves were generated using recombinant IL-22. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Table 75 summarizes levels of IL-22 measured in the supernatant. The data show that both hIL-22 are secreted at various levels from the different bacterial strains.

TABLE 75 IL-22 Secretion IL-22 Production/Secretion Dilution Corrected Genotype (ng/ml) WT 20.7 Lpp (delta lpp::CmR 87.6 expressing PhoA-IL22 from Ptet) nlpI (delta nlpI::CmR 105.4 expressing PhoA-IL22 from Ptet) tolA (delta tolA::CmR 623.2 expressing PhoA-IL22 from Ptet) PAL (delta pal::CmR 328.8 expressing PhoA-IL22 from Ptet)

Example 50 Bacterial Secretion of IL-22 and Functional Assays Generation of Bacterial Supernatant and Measurement of IL-22 Concentration

To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant was measured.

E. coli Nissle comprising a tet-inducible integrated construct (delta pal::CmR expressing PhoA-IL22 from Ptet) was grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression of hIL-22. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the supernatant was centrifuged, and filtered through a 0.22-micron filter to remove any remaining bacteria.

The concentration of hIL-22 in the cell-free medium was measured by hIL-22 ELISA (R&D Systems (DY782) ELISA for hIL-22), per manufacturer's instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Additionally, samples were diluted to ensure absence of matrix effects and to demonstrate linearity. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. The engineered bacteria comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag were determined to be secreting at 199 ng/ml supernatant.

Co-Culture Studies

To determine whether the hIL-22 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation was conducted, in which the bacterial supernatant containing secreted human IL-22 was added to the growth medium of a mammalian colonic epithelial cell line. IL-22 is known to induce the phosphorylation of STAT1 and STAT3 in Colo205 cells (see, e.g., Nagalakshmi et al., Interleukin-22 activates STAT3 and induces IL-10 by colon epithelial cells. Int Immunopharmacol. 2004 May; 4(5):679-91). Functional activity of bacterially secreted IL-22 was therefore assessed by its ability to phosphorylate STAT3 in Colo205 cells.

Colo205 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Colo205 (1e6/24 well) were serum starved overnight. Titrations of either recombinant human IL-22 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria were added to cells for 30 minutes. Cells were harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) was run in triplicate for all samples, according to manufacturer's instructions. PBS-treated cells and PBS were added as negative controls. Dilutions of samples were included to demonstrate linearity. No signal was observed for wild type Nissle. Results for the engineered strain comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag are shown in FIG. 26A, and demonstrate that hIL-22 secreted from the engineered bacteria is functionally active.

Competition Studies

As an additional control for specificity, a competition assay was performed. Titrations of anti-IL22 antibody (MAB7821, R&D Systems) were pre-incubated with constant concentrations of either rhIL22 (data not shown) or supernatants from the engineered bacteria for 15 min. Next, the supernatants/ rhIL2 solutions were added to serum-starved Colo205 cells (1e6/well) and cells were incubated for 30 min followed by pSTAT3 ELISA as described above. As shown in FIG. 26B, the phospho-Stat3 signal induced by the secreted hIL-22 is competed by the hIL-22 antibody MAB7821.

Example 51 Bacterial Secretion of GLP-1

The concentration of secreted GLP-lin the bacterial supernatant from four engineered strains comprising GLP-1 constructs/strains with different ribosome binding site (RBS) strength and two different secretion tags (PhoA or OmpF) were measured and compared.

All of the constructs were tested in a deltaLpp background. Strains are described in Table 76 (and shown in FIG. 24 and FIG. 25). 20K, 100K and 67K are numbers indicating the strengths of the RBS as determined by bioinformatics on an arbitrary scale, e.g., strength of 20K<67K<100K.

Strains were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm). Cultures were diluted to an optical density of 0.5 at which time strains were induced with ATC (100 ng/mL). After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22 micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris was spun down at 20 min at 12,000 rcf at 4.0 then placed on ice until further processing.

The concentration of GLP-lin the cell-free medium and in the bacterial cell extract was measured by Abcam kit (ab184857) according to the manufacturers protocol. Standard curves were generated using recombinant GLP-1. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Results are shown in Table 76 and FIG. 25C.

TABLE 76 GLP-1 Secretion ng/mL Strain Genotype GLP1 SYN2627 Δlpp TetR-pTet-20K RBS-PhoA-Glp1 3.6 SYN2643 Δlpp TetR-pTet-100K RBS-PhoA-Glp1 26.3 SYN2672 Δlpp TetR-pTet-20K RBS-OmpF-Glp1 2 SYN2673 Δlpp TetR-pTet-67K RBS-OmpF-Glp1 57.6

TABLE 77 Glp1 Secretion Sequences Description Sequence pTet-20K RBS-PhoA- TAATTCCTAATTTTTGTTGACACTCTATCATTGAT Glp1 AGAGTTATTTTACCACTCCCTATCAGTGATAGAG SEQ ID NO: 337 AAAAGTGAACCAAAACAGAGTCATATTTAAAGG AAGGTACAAAATGAAGCAGAGCACCATCGCGCT TGCCCTGCTGCCGTTGCTTTTCACGCCTGTCACCA AGGCTCACGATGAATTTGAGAGACATGCAGAAG GAACGTTCACATCTGATGTGTCATCATATTTGGA AGGCCAAGCTGCCAAAGAATTCATCGCATGGTTG GTGAAAGGCCGAGGATGA pTet-100K RBS- TAATTCCTAATTTTTGTTGACACTCTATCATTGAT PhoA-Glp1 AGAGTTATTTTACCACTCCCTATCAGTGATAGAG SEQ ID NO: 338 AAAAGTGAAATAAGTTTATCAAAATAAAAGGAG GTAATATATGAAGCAGAGCACCATCGCGCTTGCC CTGCTGCCGTTGCTTTTCACGCCTGTCACCAAGGC TCACGATGAATTTGAGAGACATGCAGAAGGAAC GTTCACATCTGATGTGTCATCATATTTGGAAGGC CAAGCTGCCAAAGAATTCATCGCATGGTTGGTGA AAGGCCGAGGATGA pTet-20K RBS-OmpF- TAATTCCTAATTTTTGTTGACACTCTATCATTGAT Glp1 AGAGTTATTTTACCACTCCCTATCAGTGATAGAG SEQ ID NO: 339 AAAAGTGAAGTCTTCCCGATCCTTTCCCGAGCGT ACAAAATGATGAAGCGTAACATCTTAGCCGTTAT TGTCCCCGCATTGCTTGTGGCCGGGACGGCTAAC GCACACGATGAATTTGAGAGACATGCAGAAGGA ACGTTCACATCTGATGTGTCATCATATTTGGAAG GCCAAGCTGCCAAAGAATTCATCGCATGGTTGGT GAAAGGCCGAGGATGA pTet-67K RBS OmpF- TAATTCCTAATTTTTGTTGACACTCTATCATTGAT Glp1 AGAGTTATTTTACCACTCCCTATCAGTGATAGAG SEQ ID NO: 340 AAAAGTGAAAAAACCGCCATCAAGAGTTAAGGA GGAGAATATGATGAAGCGTAACATCTTAGCCGTT ATTGTCCCCGCATTGCTTGTGGCCGGGACGGCTA ACGCACACGATGAATTTGAGAGACATGCAGAAG GAACGTTCACATCTGATGTGTCATCATATTTGGA AGGCCAAGCTGCCAAAGAATTCATCGCATGGTTG GTGAAAGGCCGAGGATGA 20K RBS CCAAAACAGAGTCATATTTAAAGGAAGGTACAA SEQ ID NO: 341 A 67K RBS AAAACCGCCATCAAGAGTTAAGGAGGAGAAT SEQ ID NO: 342 100K RBS ATAAGTTTATCAAAATAAAAGGAGGTAATAT SEQ ID NO: 343 PhoA ATGAAGCAGAGCACCATCGCGCTTGCCCTGCTGC SEQ ID NO: 344 CGTTGCTTTTCACGCCTGTCACCAAGGCT OmpF ATGATGAAGCGTAACATCTTAGCCGTTATTGTCC SEQ ID NO: 345 CCGCATTGCTTGTGGCCGGGACGGCTAACGCA Glp1 CACGATGAATTTGAGAGACATGCAGAAGGAACG SEQ ID NO: 346 TTCACATCTGATGTGTCATCATATTTGGAAGGCC AAGCTGCCAAAGAATTCATCGCATGGTTGGTGAA AGGCCGAGGATGA PhoA-Glp1 ATGAAGCAGAGCACCATCGCGCTTGCCCTGCTGC SEQ ID NO: 347 CGTTGCTTTTCACGCCTGTCACCAAGGCTCACGAT GAATTTGAGAGACATGCAGAAGGAACGTTCACAT CTGATGTGTCATCATATTTGGAAGGCCAAGCTGC CAAAGAATTCATCGCATGGTTGGTGAAAGGCCGA GGATGA OmpF-Glp1 ATGATGAAGCGTAACATCTTAGCCGTTATTGTCC SEQ ID NO: 348 CCGCATTGCTTGTGGCCGGGACGGCTAACGCACA CGATGAATTTGAGAGACATGCAGAAGGAACGTTC ACATCTGATGTGTCATCATATTTGGAAGGCCAAG CTGCCAAAGAATTCATCGCATGGTTGGTGAAAGG CCGAGGATGA Terminator AACGATTGGTAAACCCGGTGaacgcatgagAAAGCCC SEQ ID NO: 349 CCGGAAGATCACCTTCCGGGGGCTTTtttattgcgcGG ACCAAAACGAAAAAAGACGCTCGAAAGCGTCTC TTTTCTGGAATTTGGTACCGAGG

In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, and SEQ ID NO: 349.

Example 52

TABLE 78 Other Sequences of interest Wild-type clbA caaatatcacataatcttaacatatcaataaacacagtaaagtttcatgtgaaaaacat (SEQ ID NO: 350) caaacataaaatacaagctcggaatacgaatcacgctatacacattgctaacagga atgagattatctaaatgaggattgatatattaattggacatactagttatttcatcaaac cagtagagataacttccttcactatctcaatgaggaagaaataaaacgctatgatca gtttcattttgtgagtgataaagaactctatattttaagccgtatcctgctcaaaacagc actaaaaagatatcaacctgatgtctcattacaatcatggcaatttagtacgtgcaaat atggcaaaccatttatagtattcctcagttggcaaaaaagattattttaacctttcccat actatagatacagtagccgttgctattagttctcactgcgagcttggtgtcgatattga acaaataagagatttagacaactcttatctgaatatcagtcagcattatttactccaca ggaagctactaacatagtttcacttcctcgttatgaaggtcaattacttattggaaaat gtggacgctcaaagaagcttacatcaaatatcgaggtaaaggcctatctttaggact ggattgtattgaatttcatttaacaaataaaaaactaacttcaaaatatagaggttcacc tgtttatttctctcaatggaaaatatgtaactcatttctcgcattagcctctccactcatca cccctaaaataactattgagctatttcctatgcagtcccaactttatcaccacgactatc agctaattcattcgtcaaatgggcagaattgaatcgccacggataatctagacacttc tgagccgtcgataatattgattttcatattccgtcggtggtgtaagtatcccgcataatc gtgccattcacatttag clbA knock-out ggatggggggaaacatggataagttcaaagaaaaaaacccgttatctctgcgtgaaa (SEQ ID NO: 351) gacaagtattgcgcatgctggcacaaggtgatgagtactctcaaatatcacataatctt aacatatcaataaacacagtaaagtttcatgtgaaaaacatcaaacataaaatacaagc tcggaatacgaatcacgctatacacattgctaacaggaatgagattatctaaatgagga ttgaTGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATAC TTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCG GAATAGGAACTAAGGAGGATATTCATATGtcgtcaaatggg cagaattgaatcgccacggataatctagacacttctgagccgtcgataatattgattttc atattccgtcggtgg

Example 53 Assessment of In Vitro and In Vivo Activity of Biosafety System Containing Strain

The activity of the following strains is tested:

SYN-1001 comprises a construct shown in FIG. 74C knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24C (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1002 comprises a construct shown in FIG. 74C knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1003 comprises a construct shown in FIG. 74D knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24C (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1004 comprises a construct shown in FIG. 74D knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1005 comprises a construct shown in FIG. 74C knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24C (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1006 comprises a construct shown in FIG. 74C knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1007 comprises a construct shown in FIG. 74D knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1008 a construct shown in FIG. 74D knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 24D (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used.

SYN-1009 a construct shown in FIG. 74C knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive proml (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

SYN-1011 comprises a construct shown in FIG. 74D knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74A, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

SYN-1013 comprises a construct shown in FIG. 74C knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

SYN-1014 comprises a construct shown in FIG. 74D knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive proml (BBA J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 74B, except that the bla gene is replaced with the construct of FIG. 8A (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used.

TABLE 79 Biosafety System Constructs and Sequence Components SEQ ID Description Sequence NO Biosafety Plasmid ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG 352 System GGTTATTGTCTCATGAGCGGATACATATTTGAATGT Component-dap ATTTAGAAAAATAAACAAATAGGGGAATTAAAAAA A AAGCCCGCTCATTAGGCGGGCTACTACCTAGGCCG Biosafety Plasmid CGGCCGCGCGAATTCGAGCTCGGTACCCGGGGATC System Vector CTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCGG sequences, CCGCGTCGTGACTGGGAAAACCCTGGCGACTAGTC comprising dapA, TTGGACTCCTGTTGATAGATCCAGTAATGACCTCAG Kid Toxin and AACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCC R6K minimal on, GCCGGGCGTTTTTTATTGGTGAGAATCCAGGGGTCC and promoter CCAATAATTACGATTTAAATCACAGCAAACACCAC elements driving GTCGGCCCTATCAGCTGCGTGCTTTCTATGAGTCGT expression of these TGCTGCATAACTTGACAATTAACATCCGGCTCGTAG components, as GGTTTGTGGAGGGCCCAAGTTCACTTAAAAAGGAG shown in FIG. ATCAACAATGAAAGCAATTTTCGTACTGAAACATCT 74A TAATCATGCTGGGGAGGGTTTCTAATGTTCACGGGA AGTATTGTCGCGATTGTTACTCCGATGGATGAAAAA GGTAATGTCTGTCGGGCTAGCTTGAAAAAACTGATT GATTATCATGTCGCCAGCGGTACTTCGGCGATCGTT TCTGTTGGCACCACTGGCGAGTCCGCTACCTTAAAT CATGACGAACATGCTGATGTGGTGATGATGACGCT GGATCTGGCTGATGGGCGCATTCCGGTAATTGCCGG GACCGGCGCTAACGCTACTGCGGAAGCCATTAGCC TGACGCAGCGCTTCAATGACAGTGGTATCGTCGGCT GCCTGACGGTAACCCCTTACTACAATCGTCCGTCGC AAGAAGGTTTGTATCAGCATTTCAAAGCCATCGCTG AGCATACTGACCTGCCGCAAATTCTGTATAATGTGC CGTCCCGTACTGGCTGCGATCTGCTCCCGGAAACGG TGGGCCGTCTGGCGAAAGTAAAAAATATTATCGGA ATCAAAGAGGCAACAGGGAACTTAACGCGTGTAAA CCAGATCAAAGAGCTGGTTTCAGATGATTTTGTTCT GCTGAGCGGCGATGATGCGAGCGCGCTGGACTTCA TGCAATTGGGCGGTCATGGGGTTATTTCCGTTACGG CTAACGTCGCAGCGCGTGATATGGCCCAGATGTGC AAACTGGCAGCAGAAGGGCATTTTGCCGAGGCACG CGTTATTAATCAGCGTCTGATGCCATTACACAACAA ACTATTTGTCGAACCCAATCCAATCCCGGTGAAATG GGCATGTAAGGAACTGGGTCTTGTGGCGACCGATA CGCTGCGCCTGCCAATGACACCAATCACCGACAGT GGCCGTGAGACGGTCAGAGCGGCGCTTAAACATGC CGGTTTGCTGTAAGACTTTTGTCAGGTTCCTACTGT GACGACTACCACCGATAGACTGGAGTGTTGCTGCG AAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGAA GTCACCACGATTGTGCTTTACACGGAGTAGTCGGCA GTTCCTTAAGTCAGAATAGTGGACAGGCGGCCAAG AACTTCGTTCATGATAGTCTCCGGAACCCGTTCGAG TCGTTTTCCGCCCCGTGCTTTCATATCAATTGTCCGG GGTTGATCGCAACGTACAACACCTGTGGTACGTATG CCAACACCATCCAACGACACCGCAAAGCCGGCAGT GCGGGCAAAATTGCCTCCGCTGGTTACGGGCACAA CAACAGGCAGGCGGGTCACGCGATTAAAGGCCGCC GGTGTGACAATCAGCACCGGCCGCGTTCCCTGCTGC TCATGACCTGCGGTAGGATCAAGCGAGACAAGCCA GATTTCCCCTCTTTCCATCTAGTATAACTATTGTTTC TCTAGTAACATTTATTGTACAACACGAGCCCATTTT TGTCAAATAAATTTTAAATTATATCAACGTTAATAA GACGTTGTCAATAAAATTATTTTGACAAAATTGGCC GGCCGGCGCGCCGATCTGAAGATCAGCAGTTCAAC CTGTTGATAGTACGTACTAAGCTCTCATGTTTCACG TACTAAGCTCTCATGTTTAACGTACTAAGCTCTCAT GTTTAACGAACTAAACCCTCATGGCTAACGTACTAA GCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCA CGTACTAAGCTCTCATGTTTGAACAATAAAATTAAT ATAAATCAGCAACTTAAATAGCCTCTAAGGTTTTAA GTTTTATAAGAAAAAAAAGAATATATAAGGCTTTT AAAGCCTTTAAGGTTTAACGGTTGTGGACAACAAG CCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCC TCTCAAAGCAATTTTGAGTGACACAGGAACACTTA ACGGCTGACATGGGGCGCGCCCAGCTGTCTAGGGC GGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGAC AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCT Biosafety Plasmid ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG 353 System GGTTATTGTCTCATGAGCGGATACATATTTGAATGT Component- ATTTAGAAAAATAAACAAATAGGGGAATTAAAAAA ThyA AAGCCCGCTCATTAGGCGGGCTACTACCTAGGCCG Bio safety Plasmid CGGCCGCGCGAATTCGAGCTCGGTACCCGGGGATC System Vector CTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCGG sequences, CCGCGTCGTGACTGGGAAAACCCTGGCGACTAGTC comprising ThyA, TTGGACTCCTGTTGATAGATCCAGTAATGACCTCAG Kid Toxin and AACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCC R6K minimal on, GCCGGGCGTTTTTTATTGGTGAGAATCCAGGGGTCC and promoter CCAATAATTACGATTTAAATCACAGCAAACACCAC elements driving GTCGGCCCTATCAGCTGCGTGCTTTCTATGAGTCGT expression of these TGCTGCATAACTTGACAATTAATCATCCGGCTCGTA components, as GGGTTTGTGGAGGGCCCAAGTTCACTTAAAAAGGA shown in FIG. GATCAACAATGAAAGCAATTTTCGTACTGAAACAT 74B CTTAATCATGCTGGGGAGGGTTTCTAATGAAACAGT ATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGC ACACAGAAAAACGACCGTACCGGAACCGGAACGCT TTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCA AGATGGATTCCCGCTGGTGACAACTAAACGTTGCC ACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTC TTCAGGGCGACACTAACATTGCTTATCTACACGAAA ACAATGTCACCATCTGGGACGAATGGGCCGATGAA AACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGTGCCTGGCCAACGCCAGATGGTCGTCATATTGA CCAGATCACTACGGTACTGAACCAGCTGAAAAACG ACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGA ACGTAGGCGAACTGGATAAAATGGCGCTGGCACCG TGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGC AAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGAC GTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTAC GCGTTATTGGTGCATATGATGGCGCAGCAGTGCGAT CTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGAC ACGCATCTGTACAGCAACCATATGGATCAAACTCAT CTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAA GTTGATTATCAAACGTAAACCCGAATCCATCTTCGA CTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGA TCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTA AGACTTTTGTCAGGTTCCTACTGTGACGACTACCAC CGATAGACTGGAGTGTTGCTGCGAAAAAACCCCGC CGAAGCGGGGTTTTTTGCGAGAAGTCACCACGATT GTGCTTTACACGGAGTAGTCGGCAGTTCCTTAAGTC AGAATAGTGGACAGGCGGCCAAGAACTTCGTTCAT GATAGTCTCCGGAACCCGTTCGAGTCGTTTTCCGCC CCGTGCTTTCATATCAATTGTCCGGGGTTGATCGCA ACGTACAACACCTGTGGTACGTATGCCAACACCATC CAACGACACCGCAAAGCCGGCAGTGCGGGCAAAAT TGCCTCCGCTGGTTACGGGCACAACAACAGGCAGG CGGGTCACGCGATTAAAGGCCGCCGGTGTGACAAT CAGCACCGGCCGCGTTCCCTGCTGCTCATGACCTGC GGTAGGATCAAGCGAGACAAGCCAGATTTCCCCTC TTTCCATCTAGTATAACTATTGTTTCTCTAGTAACAT TTATTGTACAACACGAGCCCATTTTTGTCAAATAAA TTTTAAATTATATCAACGTTAATAAGACGTTGTCAA TAAAATTATTTTGACAAAATTGGCCGGCCGGCGCGC CGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTA CGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTC ATGTTTAACGTACTAAGCTCTCATGTTTAACGAACT AAACCCTCATGGCTAACGTACTAAGCTCTCATGGCT AACGTACTAAGCTCTCATGTTTCACGTACTAAGCTC TCATGTTTGAACAATAAAATTAATATAAATCAGCAA CTTAAATAGCCTCTAAGGTTTTAAGTTTTATAAGAA AAAAAAGAATATATAAGGCTTTTAAAGCCTTTAAG GTTTAACGGTTGTGGACAACAAGCCAGGGATGTAA CGCACTGAGAAGCCCTTAGAGCCTCTCAAAGCAAT TTTGAGTGACACAGGAACACTTAACGGCTGACATG GGGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTC CTACTCAGGAGAGCGTTCACCGACAAACAACAGAT AAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCG TTTTATTTGATGCCT Kid toxin (reverse TTAAGTCAGAATAGTGGACAGGCGGCCAAGAACTT 354 orientation) CGTTCATGATAGTCTCCGGAACCCGTTCGAGTCGTT TTCCGCCCCGTGCTTTCATATCAATTGTCCGGGGTT GATCGCAACGTACAACACCTGTGGTACGTATGCCA ACACCATCCAACGACACCGCAAAGCCGGCAGTGCG GGCAAAATTGCCTCCGCTGGTTACGGGCACAACAA CAGGCAGGCGGGTCACGCGATTAAAGGCCGCCGGT GTGACAATCAGCACCGGCCGCGTTCCCTGCTGCTCA TGACCTGCGGTAGGATCAAGCGAGACAAGCCAGAT TTCCCCTCTTTCCAT dapA ATGTTCACGGGAAGTATTGTCGCGATTGTTACTCCG 355 ATGGATGAAAAAGGTAATGTCTGTCGGGCTAGCTT GAAAAAACTGATTGATTATCATGTCGCCAGCGGTA CTTCGGCGATCGTTTCTGTTGGCACCACTGGCGAGT CCGCTACCTTAAATCATGACGAACATGCTGATGTGG TGATGATGACGCTGGATCTGGCTGATGGGCGCATTC CGGTAATTGCCGGGACCGGCGCTAACGCTACTGCG GAAGCCATTAGCCTGACGCAGCGCTTCAATGACAG TGGTATCGTCGGCTGCCTGACGGTAACCCCTTACTA CAATCGTCCGTCGCAAGAAGGTTTGTATCAGCATTT CAAAGCCATCGCTGAGCATACTGACCTGCCGCAAA TTCTGTATAATGTGCCGTCCCGTACTGGCTGCGATC TGCTCCCGGAAACGGTGGGCCGTCTGGCGAAAGTA AAAAATATTATCGGAATCAAAGAGGCAACAGGGAA CTTAACGCGTGTAAACCAGATCAAAGAGCTGGTTTC AGATGATTTTGTTCTGCTGAGCGGCGATGATGCGAG CGCGCTGGACTTCATGCAATTGGGCGGTCATGGGGT TATTTCCGTTACGGCTAACGTCGCAGCGCGTGATAT GGCCCAGATGTGCAAACTGGCAGCAGAAGGGCATT TTGCCGAGGCACGCGTTATTAATCAGCGTCTGATGC CATTACACAACAAACTATTTGTCGAACCCAATCCAA TCCCGGTGAAATGGGCATGTAAGGAACTGGGTCTT GTGGCGACCGATACGCTGCGCCTGCCAATGACACC AATCACCGACAGTGGCCGTGAGACGGTCAGAGCGG CGCTTAAACATGCCGGTTTGCTGTAA thyA ATGAAACAGTATTTAGAACTGATGCAAAAAGTGCT 356 CGACGAAGGCACACAGAAAAACGACCGTACCGGA ACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGT TTTAACCTGCAAGATGGATTCCCGCTGGTGACAACT AAACGTTGCCACCTGCGTTCCATCATCCATGAACTG CTGTGGTTTCTTCAGGGCGACACTAACATTGCTTAT CTACACGAAAACAATGTCACCATCTGGGACGAATG GGCCGATGAAAACGGCGACCTCGGGCCAGTGTATG GTAAACAGTGGCGTGCCTGGCCAACGCCAGATGGT CGTCATATTGACCAGATCACTACGGTACTGAACCAG CTGAAAAACGACCCGGATTCGCGCCGCATTATTGTT TCAGCGTGGAACGTAGGCGAACTGGATAAAATGGC GCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGT GGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCG CTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCA GCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGAC CGGTGGCGACACGCATCTGTACAGCAACCATATGG ATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTC CGCTGCCGAAGTTGATTATCAAACGTAAACCCGAA TCCATCTTCGACTACCGTTTCGAAGACTTTGAGATT GAAGGCTACGATCCGCATCCGGGCATTAAAGCGCC GGTGGCTATCTAA Kid toxin MERGEIWLVSLDPTAGHEQQGTRPVLIVTPAAFNRVT 357 polypeptide RLPVVVPVTSGGNFARTAGFAVSLDGVGIRTTGVVRC DQPRTIDMKARGGKRLERVPETIMNEVLGRLSTILT* dapA polypeptide MFTGSIVAIVTPMDEKGNVCRASLKKLIDYHVASGTS 358 AIVSVGTTGESATLNHDEHADVVMMTLDLADGRIPVI AGTGANATAEAISLTQRFNDSGIVGCLTVTPYYNRPS QEGLYQHFKAIAEHTDLPQILYNVPSRTGCDLLPETVG RLAKVKNIIGIKEATGNLTRVNQIKELVSDDFVLLSGD DASALDFMQLGGHGVISVTANVAARDMAQMCKLAA EGHFAEARVINQRLMPLHNKLFVEPNPIPVKWACKEL GLVATDTLRLPMTPITDSGRETVRAALKHAGLL ThyA polypeptide MKQYLELMQKVLDEGTQKNDRTGTGTLSIFGHQMRF 359 NLQDGFPLVTTKRCHLRSIIHELLWFLQGDTNIAYLHE NNVTIWDEWADENGDLGPVYGKQWRAWPTPDGRHI DQITTVLNQLKNDPDSRRIIVSAWNVGELDKMALAPC HAFFQFYVADGKLSCQLYQRSCDVFLGLPFNIASYAL LVHMMAQQCDLEVGDFVWTGGDTHLYSNHMDQTH LQLSREPRPLPKLIIKRKPESIFDYRFEDFEIEGYDPHPG IKAPVAI*

TABLE 80 Chromosomally Inserted Biosafety System Constructs SEQ ID Description Sequence NO Biosafety TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGGAT 360 Chromosomal CTGCTGGAACAGGTGGTGAGACTCAAGGTCATGATGGA Construct-low CGTGAACAAAAAAACGAAAATTCGCCACCGAAACGAGC copy Rep (Pi) TAAATCACACCCTGGCTCAACTTCCTTTGCCCGCAAAGC and Kis antitoxin GAGTGATGTATATGGCGCTTGCTCCCATTGATAGCAAAG (as shown in AACCTCTTGAACGAGGGCGAGTTTTCAAAATTAGGGCTG FIG. 74C) AAGACCTTGCAGCGCTCGCCAAAATCACCCCATCGCTTG CTTATCGACAATTAAAAGAGGGTGGTAAATTACTTGGTG CCAGCAAAATTTCGCTAAGAGGGGATGATATCATTGCTT TAGCTAAAGAGCTTAACCTGCTCTTTACTGCTAAAAACT CCCCTGAAGAGTTAGACCTTAACATTATTGAGTGGATAG CTTATTCAAATGATGAAGGATACTTGTCTTTAAAATTCA CCAGAACCATAGAACCATATATCTCTAGCCTTATTGGGA AAAAAAATAAATTCACAACGCAATTGTTAACGGCAAGC TTACGCTTAAGTAGCCAGTATTCATCTTCTCTTTATCAAC TTATCAGGAAGCATTACTCTAATTTTAAGAAGAAAAATT ATTTTATTATTTCCGTTGATGAGTTAAAGGAAGAGTTAA TAGCTTATACTTTTGATAAAGATGGAAATATTGAGTACA AATACCCTGACTTTCCTATTTTTAAAAGGGATGTGTTAA ATAAAGCCATTGCTGAAATTAAAAAGAAAACAGAAATA TCGTTTGTTGGCTTCACTGTTCATGAAAAAGAAGGAAGA AAAATTAGTAAGCTGAAGTTCGAATTTGTCGTTGATGAA GATGAATTTTCTGGCGATAAAGATGATGAAGCTTTTTTT ATGAATTTATCTGAAGCTGATGCAGCTTTTCTCAAGGTA TTTGATGAAACCGTACCTCCCAAAAAAGCTAAGGGGTGA GGATCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCG AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA ACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTG GGCCTTTCTGCGTTTATACCCGGGAAAAAGAGTATTGAC TtaaagtctaacctataggTATAATGTGTGGAGACCAGAGGTAAGG AGGTAACAACCATGCGAGTGTTGAAGAAACATCTTAATC ATGCTAAGGAGGTTTTCTAATGCATACCACCCGACTGAA GAGGGTTGGCGGCTCAGTTATGCTGACCGTCCCACCGGC ACTGCTGAATGCGCTGTCTCTGGGCACAGATAATGAAGT TGGCATGGTCATTGATAATGGCCGGCTGATTGTTGAGCC GTACAGACGCCCGCAATATTCACTGGCTGAGCTACTGGC ACAGTGTGATCCGAATGCTGAAATATCAGCTGAAGAAC GAGAATGGCTGGATGCACCGGCGACTGGTCAGGAGGAA ATCTGA Biosafety TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGGAT 361 Chromosomal CTTCCGGAAGACTAGGTGAGACTCAAGGTCATGATGGAC Construct- GTGAACAAAAAAACGAAAATTCGCCACCGAAACGAGCT medium copy AAATCACACCCTGGCTCAACTTCCTTTGCCCGCAAAGCG Rep (Pi) and Kis AGTGATGTATATGGCGCTTGCTCCCATTGATAGCAAAGA antitoxin (as ACCTCTTGAACGAGGGCGAGTTTTCAAAATTAGGGCTGA shown in FIG. AGACCTTGCAGCGCTCGCCAAAATCACCCCATCGCTTGC 74D) TTATCGACAATTAAAAGAGGGTGGTAAATTACTTGGTGC CAGCAAAATTTCGCTAAGAGGGGATGATATCATTGCTTT AGCTAAAGAGCTTAACCTGCTCTTTACTGCTAAAAACTC CCCTGAAGAGTTAGACCTTAACATTATTGAGTGGATAGC TTATTCAAATGATGAAGGATACTTGTCTTTAAAATTCAC CAGAACCATAGAACCATATATCTCTAGCCTTATTGGGAA AAAAAATAAATTCACAACGCAATTGTTAACGGCAAGCTT ACGCTTAAGTAGCCAGTATTCATCTTCTCTTTATCAACTT ATCAGGAAGCATTACTCTAATTTTAAGAAGAAAAATTAT TTTATTATTTCCGTTGATGAGTTAAAGGAAGAGTTAATA GCTTATACTTTTGATAAAGATGGAAATATTGAGTACAAA TACCCTGACTTTCCTATTTTTAAAAGGGATGTGTTAAATA AAGCCATTGCTGAAATTAAAAAGAAAACAGAAATATCG TTTGTTGGCTTCACTGTTCATGAAAAAGAAGGAAGAAAA ATTAGTAAGCTGAAGTTCGAATTTGTCGTTGATGAAGAT GAATTTTCTGGCGATAAAGATGATGAAGCTTTTTTTATG AATTTATCTGAAGCTGATGCAGCTTTTCTCAAGGTATTTG ATGAAACCGTACCTCCCAAAAAAGCTAAGGGGTGAGGA TCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACG CTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC CTTTCTGCGTTTATACCCGGGAAAAAGAGTATTGACTtaaa gtctaacctataggTATAATGTGTGGAGACCAGAGGTAAGGAGG TAACAACCATGCGAGTGTTGAAGAAACATCTTAATCATG CTAAGGAGGTTTTCTAATGCATACCACCCGACTGAAGAG GGTTGGCGGCTCAGTTATGCTGACCGTCCCACCGGCACT GCTGAATGCGCTGTCTCTGGGCACAGATAATGAAGTTGG CATGGTCATTGATAATGGCCGGCTGATTGTTGAGCCGTA CAGACGCCCGCAATATTCACTGGCTGAGCTACTGGCACA GTGTGATCCGAATGCTGAAATATCAGCTGAAGAACGAG AATGGCTGGATGCACCGGCGACTGGTCAGGAGGAAATC TGA Rep (Pi) TGAGACTCAAGGTCATGATGGACGTGAACAAAAAAACG 362 AAAATTCGCCACCGAAACGAGCTAAATCACACCCTGGCT CAACTTCCTTTGCCCGCAAAGCGAGTGATGTATATGGCG CTTGCTCCCATTGATAGCAAAGAACCTCTTGAACGAGGG CGAGTTTTCAAAATTAGGGCTGAAGACCTTGCAGCGCTC GCCAAAATCACCCCATCGCTTGCTTATCGACAATTAAAA GAGGGTGGTAAATTACTTGGTGCCAGCAAAATTTCGCTA AGAGGGGATGATATCATTGCTTTAGCTAAAGAGCTTAAC CTGCTCTTTACTGCTAAAAACTCCCCTGAAGAGTTAGAC CTTAACATTATTGAGTGGATAGCTTATTCAAATGATGAA GGATACTTGTCTTTAAAATTCACCAGAACCATAGAACCA TATATCTCTAGCCTTATTGGGAAAAAAAATAAATTCACA ACGCAATTGTTAACGGCAAGCTTACGCTTAAGTAGCCAG TATTCATCTTCTCTTTATCAACTTATCAGGAAGCATTACT CTAATTTTAAGAAGAAAAATTATTTTATTATTTCCGTTGA TGAGTTAAAGGAAGAGTTAATAGCTTATACTTTTGATAA AGATGGAAATATTGAGTACAAATACCCTGACTTTCCTAT TTTTAAAAGGGATGTGTTAAATAAAGCCATTGCTGAAAT TAAAAAGAAAACAGAAATATCGTTTGTTGGCTTCACTGT TCATGAAAAAGAAGGAAGAAAAATTAGTAAGCTGAAGT TCGAATTTGTCGTTGATGAAGATGAATTTTCTGGCGATA AAGATGATGAAGCTTTTTTTATGAATTTATCTGAAGCTG ATGCAGCTTTTCTCAAGGTATTTGATGAAACCGTACCTC CCAAAAAAGCTAAGGGGTGA Kis antitoxin CATACCACCCGACTGAAGAGGGTTGGCGGCTCAGTTATG 363 CTGACCGTCCCACCGGCACTGCTGAATGCGCTGTCTCTG GGCACAGATAATGAAGTTGGCATGGTCATTGATAATGGC CGGCTGATTGTTGAGCCGTACAGACGCCCGCAATATTCA CTGGCTGAGCTACTGGCACAGTGTGATCCGAATGCTGAA ATATCAGCTGAAGAACGAGAATGGCTGGATGCACCGGC GACTGGTCAGGAGGAAATCTGA RBS (low copy) GCTGGAACAGGTGG 364 RBS (medium TCCGGAAGACTAGG 365 copy) 

1. An engineered bacterium comprising a gene sequence or gene cassette for producing one or more aryl hydrocarbon receptor (AhR) agonist(s), wherein the gene sequence or gene cassette is operably linked to a directly or indirectly inducible promoter that is not associated with the gene sequence or gene cassette in nature.
 2. The engineered bacterium of claim 1, wherein the engineered bacterium comprises gene sequence for producing indole-3-acetonitrile.
 3. The engineered bacterium of claim 2, wherein the engineered bacterium comprises gene sequence encoding cyp79B2 (tryptophan N-monooxygenase).
 4. The genetically engineered bacteria of claim 2 or claim 3, wherein the engineered bacterium comprises gene sequence encoding cyp71a13 (indoleacetaldoxime dehydratase).
 5. The genetically engineered bacteria of any of claims 2-4, wherein the engineered bacterium comprises gene sequence encoding cyp79B3 (tryptophan N-monooxygenase).
 6. The genetically engineered bacteria of claim 5, wherein the cyp79B2, cyp71a13, and cyp79B3 are from Arabidopsis thaliana.
 7. The bacterium of any of claims 1-6, wherein the bacterium comprises a gene or gene cassette for producing indole-3-propionic acid.
 8. The genetically engineered bacteria of claim 7, wherein the engineered bacterium comprises gene sequence encoding tryptophan ammonia lyase.
 9. The genetically engineered bacyteris of claim 8, wherein the tryptophan ammonia lyase is from Rubrivivax benzoatilyticus.
 10. The genetically engineered bacterium of any of claims 7-9, wherein the engineered bacterium comprises one or more gene sequences encoding indole-3-acrylate reductase.
 11. The genetically engineered bacterium of claim 10, wherein the ndole-3-acrylate reductase is from Clostridum botulinum.
 12. The genetically engineered bacterium of any of claims 7-11, wherein the engineered bacterium comprises gene sequence encoding Tryptophan dehydrogenase (trpDH).
 13. The genetically engineered bacteria of claim 12, wherein the trpDH is from Nostoc punctiforme NIES-2108.
 14. The genetically engineered bacterium of any of claims 7, claim 12 and claim 13, wherein the engineered bacterium comprises gene sequence encoding fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase).
 15. The genetically engineered bacterium of claim 14, wherein the fldA is from Clostridium sporogenes.
 16. The genetically engineered bacterium of any of claims 7 and claims 12-15, wherein the bacterium comprises gene sequence(s) encoding fldB and fldC (indole-3-lactate dehydratase).
 17. The genetically engineered bacterium of claim 16, wherein the fldB and fldC is from Clostridium sporogenes.
 18. The genetically engineered bacterium of any of claims 7 and claims 12-17, wherein the engineered bacterium comprises gene sequences encoding fldD (indole-3-acrylyl-CoA reductase).
 19. The genetically engineered bacterium of claim 18, wherein the fldD is from Clostridium sporogenes.
 20. The genetically engineered bacterium of any of claims 7 and claims 12-19, wherein the engineered bacterium comprises gene sequences encoding Acul (acrylyl-CoA reductase).
 21. The genetically engineered bacteria of claim 20, wherein the Acul is from Rhodobacter sphaeroides.
 22. The genetically engineered bacterium of any of claims 7 and claims 12-21, wherein the engineered bacterium comprises gene sequence encoding fldH1 (3-lactate dehydrogenase 1).
 23. The genetically engineered bacterium of claim 22, wherein the fldH1 is from Clostridium sporogenes.
 24. The genetically engineered bacterium of any of claims 7 and claims 12-23, wherein the engineered bacterium comprises gene sequence encoding fldH2 (indole-3-lactate dehydrogenase 2).
 25. The genetically engineered bacteria of claim 24, wherein the fldH2 is from Clostridium sporogenes.
 26. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequences encoding trpDH, fldA, fldB, flD, and fldH1.
 27. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequences encoding trpDH, fldA, fldB, flD, and fldH2.
 28. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequence encoding trpDH, fldA, fldB, acuI and fldH1.
 29. The genetically engineered bacterium of claim 12, wherein the engineered bacterium comprises gene sequence encoding trpDH, fldA, fldB, acuI and fldH2.
 30. The genetically engineered bacterium of any of claims 1-29, wherein the engineered bacterium comprises gene sequence for producing tryptamine.
 31. The engineered bacteria of claim 30, wherein the engineered bacterium comprises gene sequence encoding Tryptophan decarboxylase.
 32. The engineered bacterium of claim 31, wherein the Tryptophan decarboxylase is from Catharanthus roseus.
 33. The engineered bacterium of any of claims 1-32, wherein the engineered bacterium comprises gene sequence for producing producing indole-3-acetaldehyde.
 34. The genetically engineered bacterium of claim 33, wherein the engineered bacterium comprises gene sequence encoding aro9 (L-tryptophan aminotransferase).
 35. The genetically engineered bacterium of claim 33 or claim 34, wherein the engineered bacterium comprises gene sequence encoding aspC (aspartate aminotransferase.
 36. The genetically engineered bacterium of any of claims 33-35, wherein the engineered bacterium comprises gene sequence encoding taal (L-tryptophan-pyruvate aminotransferase.
 37. The genetically engineered bacterium of any of claims 33-36, wherein the engineered bacterium comprises gene sequence encoding staO (L-tryptophan oxidase).
 38. The genetically engineered bacterium of any of claims 33-37, wherein the engineered bacterium comprises gene sequence encoding trpDH (Tryptophan dehydrogenase).
 39. The genetically engineered bacterium of any of claims 33-38, wherein the engineered bacterium comprises gene sequence encoding ipdC (Indole-3-pyruvate decarboxylase).
 40. The genetically engineered bacterium of claim 33, wherein the engineered bacterium comprises gene sequence encoding tdc (Tryptophan decarboxylase).
 41. The genetically engineered bacterium of claim 33 or claim 40, wherein the engineered bacterium comprises gene sequence encoding tynA (Monoamine oxidase).
 42. The genetically engineered bacterium of any of claims 1-41, wherein the engineered bacterium comprises gene sequence for producing indole-3-acetic acid.
 43. The genetically engineered bacterium of claim 42, wherein the bacterium comprises gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase), aspC (aspartate aminotransferase), taal (L-tryptophan-pyruvate aminotransferase), staO (L-tryptophan oxidase), trpDH (Tryptophan dehydrogenase), iad1 (Indole-3-acetaldehyde dehydrogenase), AAO1 (Indole-3-acetaldehyde oxidase), ipdC (Indole-3-pyruvate decarboxylase), ipdC (Indole-3-pyruvate decarboxylase), tdc (Tryptophan decarboxylase), tynA (Monoamine oxidase), yuc2 (indole-3-pyruvate monooxygenase), IaaM (Tryptophan 2-monooxygenase), and iaaH (Indoleacetamide hydrolase).
 44. The genetically engineered bacterium of any of claims 1-43, wherein the bacterium further comprises gene sequence for producing tryptophan.
 45. The genetically engineered bacterium of any of claims 1-44, wherein the bacterium further comprises gene sequence encoding one or more tryptophan transporters.
 46. The genetically engineered bacterium of claim 45, wherein the tryptophan transporter is selected from mtr, aroP, and tnaB.
 47. The bacterium of any of claims 1-46, wherein the bacterium further comprises gene sequence for producing kynurenine.
 48. The bacterium of any of claims 1-47, wherein the bacterium further comprises a gene sequence for producing kynurenic acid.
 49. The bacterium of any of claims 1-48, wherein the bacterium further comprises a gene sequence for producing an indole.
 50. The genetically engineered bacterium of any of claims 1-49, wherein the bacterium further comprises gene sequence encoding a non-native metabolic or satiety effector molecule.
 51. The bacterium of claim 50, wherein the metabolic or satiety effector molecule is selected from a a short-chain fatty acid, butyrate, propionate, acetate, GLP-1, IL-22, IL-10, bile salt hydrolase, n-acyl-phophatidylethanolamine (NAPE), a n-acyl-ethanolamines (NAE), a ghrelin receptor antagonist, peptide YY3-36, a cholecystokinin (CCK), CCK58, CCK33, CCK22, CCK8, a bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, entero statin, oxyntomodulin, pancreatic peptide, a serotonin receptor agonist, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN),nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA).
 52. The bacterium of claim 51, wherein the metabolic or satiety effector molecule is a short-chain fatty acid.
 53. The bacterium of claim 52, wherein the metabolic or satiety effector molecule is butyrate.
 54. The bacterium of claim 52, wherein the metabolic or satiety effector molecule is propionate.
 55. The bacterium of claim 52, wherein the metabolic or satiety effector molecule is GLP1.
 56. The bacterium of any of claims 1-55, wherein the gene sequence is operably linked to a directly or indirectly inducible promoter that is induced by exogenous environmental conditions.
 57. The bacterium of claim 56, wherein the promoter is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut.
 58. The bacterium claim 57, wherein the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions.
 59. The bacterium of claim 58, wherein the promoter is selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter.
 60. The bacterium of claim 59, wherein the promoter is a FNR-inducible promoter.
 61. The bacterium of any of claims 1-57, wherein the promoter is regulated by a reactive nitrogen species (RNS).
 62. The bacterium of any of claims 1-57, wherein the promoter is regulated by a reactive oxygen species (ROS).
 63. The bacterium of any one of claims 1-62, wherein the gene sequence and operatively linked promoter are present on a plasmid in the bacterium.
 64. The bacterium of any one of claims 1-62, wherein the gene sequence and operatively linked promoter are present on a chromosome in the bacterium.
 65. The bacterium of any one of claims 1-64, wherein the bacterium is an auxotroph comprising a deletion or mutation in a gene required for cell survival and/or growth.
 66. The genetically engineered bacterium of claim 65, wherein the bacterium is an auxotroph in diaminopimelic acid or an enzyme in the thymidine biosynthetic pathway.
 67. The bacterium of any one of claims 1-66, wherein the bacterium comprises a kill switch.
 68. The bacterium of any of claims 1-67, wherein the bacterium is a non-pathogenic bacterium.
 69. The bacterium of claim 68, wherein the bacterium is a probiotic or a commensal bacterium.
 70. The bacterium of claim 69, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.
 71. The bacterium of claim 70, wherein the bacterium is Escherichia coli strain Nissle.
 72. A pharmaceutically acceptable composition comprising the bacterium of any one of claims 1-71; and a pharmaceutically acceptable carrier.
 73. The pharmaceutically acceptable composition of claim 72, wherein the composition is formulated for oral or rectal administration.
 74. A method of treating a metabolic disease in a subject in need thereof comprising the step of administering to the subject the composition of claim 72 or claim
 73. 75. The method of claim 74, wherein the disorder of condition is selected from the group consisting of: type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome. 